Methods of treating hepatic encephalopathy

ABSTRACT

The application describes treatment of hepatic encephalopathy using gastrointestinal specific antibiotics. One example of a gastrointestinal specific antibiotic is rifaximin. The instant application also provides methods for determining if a subject has a neurological condition or hepatic encephalopathy by determining the critical flicker frequency and/or the venous ammonia level of the subject at two or more time points. The invention further provides methods for treating these subjects.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/391,438, filed Apr. 23, 2019, which is a continuation of U.S.application Ser. No. 15/824,209, filed Nov. 28, 2017, which is acontinuation of U.S. application Ser. No. 15/212,720, filed Jul. 18,2016, which is a continuation of U.S. application Ser. No. 13/914,074,filed Jun. 10, 2013, which is a continuation of U.S. application Ser.No. 12/964,847 filed Dec. 10, 2010, which is a continuation of U.S.application Ser. No. 12/792,658, filed Jun. 2, 2010, which is acontinuation-in-part of U.S. application Ser. No. 12/572,344, filed Oct.2, 2009 which claims the benefit of U.S. Provisional Application Ser.No. 61/102,349, filed Oct. 2, 2008. U.S. application Ser. No. 12/792,658also claims the benefit of U.S. Provisional Application No. 61/183,513filed Jun. 2, 2009; U.S. Provisional Application No. 61/262,525, filedNov. 18, 2009; U.S. Provisional Application No. 61/305,854, filed Feb.18, 2010; U.S. Provisional Application No. 61/306,935, filed Feb. 22,2010; U.S. Provisional Application No. 61/307,417, filed Feb. 23, 2010;and U.S. Provisional Application No. 61/316,796, filed Mar. 23, 2010.The entire contents of each of the aforementioned applications is herebyexpressly incorporated herein by reference.

BACKGROUND

Hepatic encephalopathy (HE) is caused by a reversible decrease inneurologic function associated with liver failure and portosystemicvenous shunting. HE occurs in 1 of every 3 cases of cirrhosis, in casesof fulminant hepatic failure reported in the United States (US), and ispresent in nearly half of patients reaching end-stage liver disease. Itmay occur at any age, but the peaks parallel those of fulminant liverdisease (peak=40's), and cirrhosis (peak=late 50's).

The incidence of HE is likely to increase with the incidence ofhepatitis C in the general population and cirrhotics in aging patients.Acute HE signifies a serious prognosis with a 40% likelihood of survivalfor 1 year. There is a need in the art for a compositions and methodsfor treating and preventing HE.

Moreover, methods of predicting a breakthrough HE event or fordetermining when to prophylactically treat a subject prior to theoccurrence of a breakthrough event are necessary in the art.

SUMMARY

Provided herein are compositions and methods for the prevention andtreatment of hepatic encephalopathy.

One embodiment is a method of treating or preventing hepaticencephalopathy (HE) in a subject comprising administering to a subject agastrointestinal (GI) specific antibiotic. In one embodiment the GIspecific antibiotic is rifaximin. In another embodiment, the rifaximinis 1100 mg/day of rifaximin.

Another embodiment is a method of decreasing a subject's risk of ahepatic encephalopathy HE breakthrough episode by administering a GIspecific antibiotic to a subject suffering from HE.

Yet another embodiment is a method of maintaining remission of hepaticencephalopathy in a subject by administering a GI specific antibiotic toa subject suffering from HE.

Still another embodiment is a method of reducing the frequency ofhospitalization visits by an HE patient, comprising administering a GIspecific antibiotic to a subject suffering from HE.

In one embodiment, the GI specific antibiotic is administered to thesubject with lactulose, prior to treatment with lactulose, or followingtreatment with lactulose. In one embodiment the subject or a health careworker is advised to administer the GI specific antibiotic withlactulose.

In one embodiment the subject or a health care worker is advised by apharmaceutical label or insert to administer the GI specific antibioticwith lactulose in order to maintain remission of HE, or to decrease therisk for episodes of overt HE.

In one embodiment, the subject or health care worker is advised toadminister two 550 mg tablets of rifaximin twice daily with lactulose.Lactulose use may be titrated over time so that the subject maintains2-3 soft stool bowel movements per day. In one embodiment the lactuloseis administered in 15 ml dosages, wherein each 15 ml dosage contains 10mg of lactulose.

In one embodiment, subjects in need of treatment for HE and having aChild-Pugh grade of A or B are treated with a GI specific antibiotic.

In another embodiment, subjects in need of treatment for HE having aChild-Pugh grade of A or B are treated with a GI specific antibiotic incombination with lactulose.

In another embodiment, subjects having a Child-Pugh grade of A or B, ortheir health care worker, are advised that they should be treated with aGI specific antibiotic. The advice can be oral or written advice, suchas on a pharmaceutical label or package insert.

In another embodiment, subjects having a Child-Pugh grade of A or B, ortheir health care worker, are advised that they should be treated with aGI specific antibiotic in combination with lactulose.

In one embodiment, a subject in need of treatment for HE and having aChild-Pugh grade of less than C is treated with a GI specificantibiotic. In one embodiment, a subject in need of treatment for HE andhaving a Child-Pugh grade of less than C is treated with a GI specificantibiotic and lactulose.

In another embodiment, a subject in need of treatment for HE, or theirhealth care worker is advised of the risk for anaphylaxis prior totreatment with a GI specific antibiotic.

In one embodiment, subjects in need of treatment for HE and having aMELD score of 25 or less are treated with a GI specific antibiotic.

In another embodiment, subjects in need of treatment for HE having aMELD score of 25 or less are treated with a GI specific antibiotic incombination with lactulose.

In another embodiment, subjects having a MELD score of 25 or less areadvised that they should be treated with a GI specific antibiotic. Theadvice can be oral or written advise, such as on a pharmaceutical labelor package insert.

In another embodiment, subjects having a MELD score of 25 or less areadvised that they should be treated with a GI specific antibiotic incombination with lactulose.

The novel methods and devices presented herein are based in part on thefinding of a correlation between CFF and/or venous ammonia concentrationand the occurrence of breakthrough HE events. Moreover, the novelmethods and devices are also based on the discovery that the timeweighted average CFF and/or venous ammonia concentration is an accuratepredictor of breakthrough HE events and prognosis of subjects with HE.

Accordingly, in one aspect provided herein are methods of diagnosinghepatic encephalopathy (HE) in a subject by determining the criticalflicker frequency (CFF) of a subject at two or more time points, whereina decrease in the CFF is indicative that the subject has hepaticencephalopathy. In a related embodiment, the CFF comprises the CFF timeweighted average. In another related embodiment, the CFF time weightedaverage comprises less than about 24 Hz.

In another aspect, provided herein are methods of determining asubject's risk of an HE breakthrough event, by determining the criticalflicker frequency (CFF) of a subject at two or more time points, whereina decrease in the CFF is indicative that the subject has an increasedrisk of a HE breakthrough event. In a related embodiment, the CFFcomprises the CFF time weighted average. In another related embodiment,the CFF time weighted average comprises less than about 24 Hz. Inanother embodiment, a CFF time weighted average of 10 Hz is indicativethat the subject has the greatest chance of a HE breakthrough event.

In another aspect, provided herein are methods of determining theprognosis of a subject having HE, by determining the critical flickerfrequency (CFF) of a subject at two or more time points, wherein adecrease in the CFF is indicative that the subject has a poor prognosis.In a related embodiment, the CFF comprises the CFF time weightedaverage. In another related embodiment, the CFF time weighted averagecomprises less than about 24 Hz, or less than about 20 Hz.

In another aspect, provided herein are methods of treating or preventingan HE event, by determining the critical flicker frequency (CFF) of asubject at two or more time points, administering to a subject having adecrease in the CFF average between the time points an effective amountof a GI specific antibiotic, (e.g., rifaximin), thereby treating orpreventing an HE event. In another embodiment, the subject isadministered a GI specific antibiotic when the CFF time weighted averageis less than 20 Hz.

In one embodiment, the subject is also administered lactulose. Inanother embodiment, the risk is increased as compared to a controlsubject without HE.

In related embodiments, the two or more time points occur within oneweek, four weeks, six months, or more.

In another aspect provided herein are methods of diagnosing hepaticencephalopathy (HE) in a subject by determining the venous ammonialevels of a subject at two or more time points, wherein an increase inthe venous ammonia levels is indicative that the subject has hepaticencephalopathy.

In one embodiment, the venous ammonia level comprises the time weightedaverage venous ammonia levels. In another embodiment, the time weightedaverage venous ammonia level comprises more than about 100 μmol/L.

In another aspect, provided herein are methods of determining asubject's risk of an HE breakthrough event, by determining the venousammonia levels of a subject at two or more time points, wherein anincrease in the venous ammonia levels is indicative that the subject hasan increased risk of a HE breakthrough event.

In one embodiment, the venous ammonia level comprises the time weightedaverage venous ammonia levels. In another embodiment, the time weightedaverage venous ammonia level comprises more than about 100 μmol/L.

In another aspect, provided herein are methods of determining theprognosis of a subject having HE, by determining the venous ammonialevel of a subject at two or more time points, wherein an increase inthe venous ammonia level is indicative that the subject has a poorprognosis.

In one embodiment, the venous ammonia level comprises the time weightedaverage venous ammonia levels. In another embodiment, the time weightedaverage venous ammonia level comprises more than about 100 μmol/L, ormore than about 110 μmol/L.

In another aspect, provided herein are methods of treating or preventingan HE event, by determining the venous ammonia level of a subject at twoor more time points, administering to a subject having an increase inthe venous ammonia level between the time points an effective amount ofRifaximin, thereby treating or preventing an HE event.

In one embodiment, the venous ammonia level comprises the time weightedaverage venous ammonia levels. In another embodiment, the time weightedaverage venous ammonia level comprises more than about 100 μmol/L.

In another embodiment, the methods further comprise administeringlactulose.

In related embodiments, the two or more time points occur within oneweek, two weeks, three weeks, four weeks, five weeks, six weeks, eightweeks, six months, or more. Also included are time points at any time inbetween the points.

In another aspect, provided herein are methods of treating a subjecthaving HE comprising administering to the subject an effective amount ofa GI specific antibiotic, e.g., rifaximin, thereby treating the subject.

In related embodiments, the GI specific antibiotic, e.g., rifaximin, isadministered for greater than 365 days, greater than 730 days, greaterthan 1095 days, or for the remainder of the life of the subject.

In another aspect provided herein are methods of diagnosing aneurological disease in a subject, by determining the critical flickerfrequency (CFF) of a subject at two or more time points, wherein adecrease in the CFF is indicative that the subject has a neurologicaldisease. The decrease may be between any two time points. For example,between the first and second time points, between the first and thirdtime points, between the first and fourth time points, between thesecond and third time points, between the second and fourth time pointsand the like.

In a related embodiment, the CFF comprises the CFF time weightedaverage. In another embodiment, the neurological disease or disordercomprises Alzheimer's disease or Parkinson's disease. Also included arediseases in which the CFF is modulated in a subject. Modulated, as usedherein includes increases or decreasing from a baseline or a change fromany measurement, even if not baseline for the subject. The baseline fora subject may be determined when a subject not experiencing an HE eventor other neurological event, when a subject has recovered or is inremission from an HE event or other neurological event.

In another aspect, provided herein are methods of determining theprognosis of a subject having a neurological disease, by determining thecritical flicker frequency (CFF) of a subject at two or more timepoints, wherein a decrease in the CFF is indicative that the subject hasa poor prognosis. Poor prognosis, as used herein, includes a prognosisthat life expectancy is less than prior prognosis.

In a related embodiment, the CFF comprises the CFF time weightedaverage. In another embodiment, the neurological disease or disordercomprises Alzheimer's disease, Parkinson's disease, trauma, migraine,chronic headache, insomnia and other sleep disorders, and/or epilepsy.

In related embodiments, the two or more time points occur within oneweek, four weeks, six months, or more.

In another aspect, provided herein are computerized methods foridentifying subjects having a neurological disease by maintaining adatabase of CFFs for subjects at various time points and stages ofdisease progression, comparing the results of an individuals CFF resultstaken at two or more time points to the database, obtaining thediagnosis of a neurological disease from the computer if subject has CFFresults that decrease between measurements.

In one embodiment, the CFF comprises the CFF time weighted average. Inanother embodiment the CFF time weighted average or a second orsubsequent time point comprises less than about 24 Hz. In anotherembodiment, the methods further comprise controlling a printing deviceto print a report based on the results of the method.

In another aspect, provided herein are business methods for decreasinghealthcare costs, comprising determining the critical flicker frequency(CFF) of a subject at two or more time points, storing patientinformation on a computer processor, determining if the subject has aneurological disease by determining if the CFF value has decreasedbetween time points, and treating the subject as necessary to avoid ordelay hospitalization.

In another aspect, provided herein are business methods for decreasinghealthcare costs comprising determining the critical flicker frequency(CFF) of a subject at two or more time points, storing patientinformation on a computer processor, determining if the subject hashepatic encephalopathy by determining if the CFF value has decreasedbetween time points, treating the subject as necessary to avoid or delayhospitalization.

A device for determining the risk of an HE event, comprising, a “flickerbox,” a measurement device to CFF, and a computer with an algorithm. Inanother embodiment, the method further comprises a memory device capableof storing CFF data.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a line graph comparing lactulose daily use between subjectstaking placebos and subjects taking rifaximin.

FIG. 2 is a line graph showing Kaplan Meier estimates of thedistribution of time to a breakthrough HE event.

FIG. 3 is a line graph showing Kaplan Meier estimates of thedistribution of time to a first HE related hospitalization.

FIG. 4 is a line graph showing Kaplan Meier estimates of thedistribution of time to a first increase in Conn scores.

FIG. 5 is a line graph showing Kaplan Meier estimates of thedistribution of time to a first increase in an Asterixis grade.

FIG. 6 depicts the time to first breakthrough overt HE episode (up to 6months of treatment, day 170 in the first study) (ITT Population).

FIG. 7 is a comparison of time to first breakthrough overt HE episode inthe first study (rifaximin versus placebo groups) and the second study(new to rifaximin group).

FIG. 8 depicts a comparison of time to first breakthrough overt heepisode during placebo experience (the first study) and after crossoverto rifaximin experience (the second study) among the first study placebosubjects who started rifaximin in the second study.

FIG. 9 depicts the time to first HE-related hospitalization (up to 6months of treatment, day 170, in the first study).

FIG. 10 depicts the time to first HE-caused hospitalization in the firststudy (ITT population).

FIG. 11 depicts the time to First Increase in Conn Score (up to 6 monthsof treatment, day 170, the first study) (ITT Population).

FIG. 12 depicts the time to first Increase in asterixis grade (up to 6months of treatment, day 170, the first study) (ITT Population).

FIG. 13 depicts the Kaplan Meier estimates of distribution of time tofirst breakthrough HE for continuing rifaximin subjects who did not havean HE episode in the first study vs placebo.

FIGS. 14A-B depict CLDQ results, as measured by Twa, between therifaximin and placebo groups in the frequency distributions of Twascores for the fatigue domain and overall domain.

FIG. 15 shows the pathogenesis of HE.

FIG. 16 shows the clinical presentation of HE. The classification was bythe 1998 WCOG Working Group. Adapted from Ferenci P, et al. Hepatology.2002; 35:716-721.

FIG. 17 shows the HESA adaptation of Conn Score.

FIG. 18 shows the impact of HE on the patient and caregiver.

FIG. 19 depicts HE hospitalizations and economic impact.

FIG. 20 shows the influence of Liver Impairment on Rifaximin PK.

FIG. 21 demonstrates that rifaximin exposure is significantly lower thanother antibiotic exposures. Well et al., Int J Antimicrob Agents 10(1998) 31-38. In patients with greatest liver impairment, rifaximinexposure is >200-fold lower than rifampin exposure; >35-fold lower thannorfloxacin exposure; and ≥10-fold lower than neomycin exposure.

FIG. 22 shows drug interactions with midazolam and rifaximin. Nosignificant inhibition of CYP enzymes, P-glycoprotein, or BSEP.Portosystemic shunting in liver impairment may reduce liver exposure.

FIG. 23 shows the effect on blood ammonia. Rifaximin 1200 mg/day for 5-10 days decreased blood ammonia (p<0.0001). Corresponding improvementin HE grade (p<0.0001), neurological, neuropsychiatric, and psychometricparameters. Correlation between ammonia reduction over time and HE wasexamined.

FIG. 24 depicts the Kaplan-Meier Event-Free Curves in HE Study (Time toFirst Breakthrough-HE Episode up to 6 Months of Treatment, Day 170) (ITTPopulation).

FIG. 25 depicts Kaplan-Meier Event-Free Curves in Pivotal HE Study (Timeto First HE-Related Hospitalization in HE Study up to 6 Months ofTreatment, Day 170) (ITT Population).

FIG. 26 is a line graph showing the time to First Breakthrough HEEpisode.

FIG. 27 is a chart showing hazard ratios for the risk of experiencingbreakthrough overt HE (rifaximin group divided by placebo group) foreach subgroup.

FIG. 28 is a line graph illustrating a time to First HE-RelatedHospitalization.

FIG. 29 is a bar chart illustrating that lactulose use between a controlgroup and a group taking rifaximin was the same.

FIG. 30 is a chart illustrating that there was a consistency oftreatment affect across various subgroups that were administeredrifaximin.

FIG. 31 depicts the distribution of time-weighted average CFF results bybreakthrough overt HE status.

FIG. 32 depicts receiver operating characteristic curve for CFF resultsin the prediction of breakthrough overt HE.

FIG. 33 depicts the distribution of time-weighted average venous ammoniaconcentrations results by breakthrough overt HE status.

FIG. 34 depicts receiver operating characteristic curve for venousammonia levels in the prediction of breakthrough overt HE.

DETAILED DESCRIPTION

Hepatic encephalopathy, also known as hepatic coma or portal-systemicencephalopathy (PSE), is a serious, rare, complex, episodic,neuropsychiatric syndrome associated with advanced liver disease.Hepatic encephalopathy is a formidable burden on the patient, his/herfamily, and the healthcare system; and the current standard of care isinadequate. Overt, episodic HE is common among patients with livercirrhosis. The condition is rare among individuals in the overall,general population. Overt HE episodes are debilitating, can presentwithout warning, render the patient incapable of self-care, andfrequently result in hospitalization. The frequency of hospitalizationsdue to HE increased since 1993 to over 40,000 patients in 2003; and in2004, 50,962 patients were hospitalized with a principal diagnosis ofHE.

The main pathogenesis of HE is related to nitrogenous substances derivedfrom the gut adversely affecting brain function. The most influential ofthese compounds is thought to be ammonia, a byproduct of proteindigestion that is normally detoxified by the liver. Correlation of bloodlevels with mental state in cirrhosis, however, is inaccurate, in part,because the blood-brain barrier permeability to ammonia is increased inpatients with HE. Other gut-derived toxins have also been proposed asbeing responsible for HE.

In patients with chronic liver disease, the occurrence of hepaticencephalopathy is associated with a low quality of life compared toage-matched patients without HE. Overt HE episodes are debilitating, canpresent without warning, render the patient incapable of self-care, andfrequently result in hospitalization. Patients with HE experiencesymptoms including fatigue, daytime sleepiness, and lack of awareness(Conn score 1); and confusion and disorientation (Conn score 2) thatsignificantly interfere with day-to-day function and decreased abilityfor self care. Often, this lack of self care leads to improper nutritionand non-adherence to therapy and further escalates into more severesymptoms such as increased somnolence, gross disorientation and stupor(Conn score 3) or coma (Conn score 4).

A history of overt HE episodes and the severity of HE episodes were alsofound to be predictive of decreased survival in patients with chronicliver disease. In patients with liver cirrhosis and a history of overtHE episodes, survival probability was 42% at 1 year and 23% at 3 yearsafter experiencing an HE episode. In another analysis, the occurrence ofan HE episode of Conn score 2 in patients with cirrhosis was associatedwith a 4-fold increase in the risk of death.

The inventors of the instant application have determined that there is acorrelation between CFF and venous ammonia concentration and theoccurrence of breakthrough HE events. Moreover, the inventors havedetermined that time weighted average CFF or venous ammoniaconcentration is an accurate predictor of breakthrough HE events andprognosis of subjects with HE. In another embodiment, the inventors havedetermined that subjects who continue taking Rifaximin for a longduration of time, e.g., greater than 1.5 years, continue to seebeneficial results, e.g., decreased incidence of breakthrough HE events.

In certain embodiments, provided herein are methods for determining if asubject has a neurological disease or HE. The methods of the inventionrely on determining the critical flicker frequency or the venous ammonialevel.

Critical flicker frequency, also called CFF, can be determined, forexample, by standard methods known in the art. Moreover, commercialinstruments are available to measure CFF, which are known by thoseskilled in the art.

Critical flicker frequency tests utilize, for example, the correlationbetween cerebral processing of oscillatory visual stimuli and CNSimpairment due to increased HE severity. This test identifies afrequency at which a flickering light is perceived by a subject as asteady light. A decline in this frequency has been associated withincreasing severity of HE. In one example, circular light pulses with a1:1 ratio between the visual impulse and the interval were used withdecreasing frequency in gradual steps of 0.5 to 0.1 Hz/second. Thefrequency of the white light, which is initially generated as ahigh-frequency pulse (50 Hz) and which gives the patient the impressionof a steady light, can be reduced gradually until the subject had theimpression that the steady light had changed to a flicker. The subjectregistered this change by pressing a hand-held switch. The flickerfrequencies can be measured multiple times and the mean values for eachsubject can be calculated.

In some embodiments, CFF values are tracked over time for each subject.From these values the area under the CFF versus time curve (AUC) couldbe calculated using calculations that are standard in the art. Forexample, AUC can be calculated using the trapezoidal rule. To use thetrapezoidal rule, data points are connected by straight line segments,perpendiculars are erected from the abscissa to each data point, and thesum of the areas of the triangles and trapezoids so constructed iscomputed and equals the AUC.

To accurately describe the variation in the CFF over time for eachsubject the time-weighted average (twa) can be computed. To calculatedthe twa, the results of the CFF test over time or the venous ammonialevels are expressed as:

${{twa} = \frac{AUC}{T}},$

where T is the exposure time. Thus, twa describes the average CFF and/orvenous ammonia level effect between multiple time points.

The correlation between twa and the presence or absence of breakthroughHE episode can be analyzed with analysis of variance and Spearman rankcorrelation coefficient. Additionally, a ROC curve analysis can beperformed to evaluate the accuracy of the twa to discriminate betweenthe presence or absence of breakthrough episodes. A ROC analysis for thedata collected in the Examples demonstrated that the methodology is ahighly accurate predictor of HE.

These toxic compounds gain access to the systemic circulation as aresult of decreased hepatic function or portal-systemic shunts. Once inbrain tissue, the compounds produce alterations of neurotransmissionthat affect consciousness and behavior. HE is attributed to globalcentral nervous system depression from nitrogenous compounds that resultin excitation of gamma-aminobutyric acid (GABA) and decreasedneurotransmission of glutamate.

Precipitating factors include azotemia (29%), sedatives, tranquilizers,analgesics (24%), gastrointestinal bleeding (18%), excess dietaryprotein (9%), metabolic alkalosis (11%), infection (3%), constipation(3%). Surgery, particularly transjugular intrahepatic portal-systemicshunt (TIPS) procedures, also may precipitate HE. HE due to unknowncauses accounts for only 2% of cases.

Initial manifestations are subclinical and require psychometric testingfor diagnosis. There are 4 progressive stages of impairment known as theWest Haven criteria (or Conn score) which range from Stage 0 (Lack ofdetectable changes in personality) to Stage 4 (Coma, decerebrateposturing, dilated pupils) as discussed in more detail below.

HE is manifested as a continuum of psychomotor dysfunction, impairedmemory, increased reaction time, sensory abnormalities, poorconcentration and in severe forms, as coma. Changes may be observed inpersonality, consciousness, behavior and neuromuscular function.Neurologic signs may include hyperreflexia, rigidity, myoclonus andasterixis (coarse “flapping” muscle tremor). Cognitive tasks such asconnecting numbers with lines can be abnormal. Fetor hepaticus (sweetbreath odor) may be present. Electroencephalogram (EEG) tracings shownonspecific slow, triphasic wave activity mainly over the frontal areas.Prothrombin time may be prolonged and not correctable with Vitamin K. Acomputed tomography scan of the head may be normal or show generalatrophy. Finally, signs of liver disease such as jaundice and ascitesmay be noted.

Diagnosis of HE is made on the basis of medical history, and physicaland mental status examinations with the required clinical elements beingknowledge of existent liver disease, precipitating factor(s), and/orprior history of HE. An EEG may show slow-wave activity, even in mildcases. An elevated serum ammonia level is characteristic but notessential, and correlates poorly with the level of encephalopathy

Management of patients with chronic HE includes 1) provision ofsupportive care, 2) identification and removal of precipitating factors,3) reduction of nitrogenous load from the gut, and 4) assessment of theneed for long term therapy. The nitrogenous load from the gut istypically reduced using non-absorbable disaccharide (lactulose) and/orantibiotics.

Lactulose is considered a first-line treatment in the United States.Lactulose is metabolized by the intestinal bacteria of the colon, whichleads to reduced fecal pH, then to a laxative effect, and finally tofecal elimination. The reduced fecal pH ionizes ammonia (NH₃) to theammonium ion (NH₄ ⁺) which is used by the bacteria for amino acid andprotein synthesis. This lowers the serum ammonia levels and improvesmental function.

Conventional therapy aims to lower the production and absorption ofammonia. Lactulose is typically used in doses of 30-60 g daily. However,the dose can be titrated up to 20-40 g TID-QID to affect 2-3 semi-formedbowel movements per day. If lactulose cannot be administered orally orper nasogastric tube, for example to patients with stage 3 and 4 HE, itmay be given as a 300 cc (200 g) retention enema.

For acute encephalopathy, lactulose can be administered either orally,by mouth or through a nasogastric tube, or via retention enemas. Theusual oral dose is 30 g followed by dosing every 1 to 2 hours untilevacuation occurs. At that point, dosing is adjusted to attain two orthree soft bowel movements daily.

Lactulose for is readily available over-the-counter. A convenient andrelatively tasteless formulation, often referred to in the trade as“lactulose powder for oral solution” can be obtained, for example, fromBertek Pharmaceuticals, Sugarland, Tex. as Kristalose® in 10 and 20 gmpackets. The lactulose syrups commonly sold as laxatives includeCephulac®, Chronulac®, Cholac®, and Enulose®. These syrups can besubstituted for lactulose powder by using sufficient syrup to providethe desired dosage of lactulose; typically, the named syrups containabout 10 gm lactulose in 15 ml of syrup.

Broad-spectrum, GI-active antibiotics including neomycin, metronidazole,vancomycin and paromomycin have been used with or without lactulose.Current guidelines recommend neomycin at 1 to 2 g/day by mouth withperiodic renal and annual auditory monitoring or metronidazole at 250.Lactulose can induce diarrhea leading to dehydration, a precipitatingfactor of HE. Additionally, compliance with lactulose is limited bypatient dislike of its overly sweet taste. In addition, a dosingschedule that is linked to bowel habits and side effects of flatulence,bloating, diarrhea (which leads to dehydration), and acidosis makelactulose difficult to use long-term.

Antibiotic use in treatment of HE is hampered by toxicity associatedwith long-term use. Specifically, systemic absorption of neomycin,metronidazole and ampicillin has led to rare cases of nephrotoxicity,ototoxicity, S. enterocolitis, and/or development of resistant bacterialstrains. Additionally, neomycin inhibits only aerobic bacteria.Metronidazole is metabolized slowly in patients with hepaticdysfunction, has a potential for alcohol interactions (disulfiram-likeeffect), and high blood levels may result in seizures.

One gastrointestinal specific antibiotic is rifaximin. Rifaximin is anonaminoglycoside, semisynthetic antibiotic derived from rifamycin O. Itis a non-systemic, non-absorbed, broad-spectrum, oral antibioticspecific for enteric pathogens of the GI tract. Rifaximin was found tobe advantageous in treatment of HE relative to previously usedantibiotics; e.g., negligible systemic absorption (<0.4%) regardless offood intake or presence of GI disease and exhibits no plasmaaccumulation with high or repeat doses. The lack of systemic absorptionmakes rifaximin safe and well tolerated, thus improving patientcompliance and reducing side effects associated with currently knowntreatments.

Rifaximin (INN; see The Merck Index, XIII Ed., 8304) is an antibioticbelonging to the rifamycin class of antibiotics, e.g., a pyrido-imidazorifamycin. Rifaximin exerts its broad antibacterial activity, forexample, in the gastrointestinal tract against localizedgastrointestinal bacteria that cause infectious diarrhea, irritablebowel syndrome, small intestinal bacterial overgrowth, Crohn's disease,and/or pancreatic insufficiency. It has been reported that rifaximin ischaracterized by a negligible systemic absorption, due to its chemicaland physical characteristics (Descombe J. J. et al. Pharmacokineticstudy of rifaximin after oral administration in healthy volunteers. IntJ Clin Pharmacol Res, 14 (2), 51-56, (1994)).

Rifaximin is described in Italian Patent IT 1154655 and EP 0161534. EPpatent 0161534 discloses a process for rifaximin production usingrifamycin O as the starting material (The Merck Index, XIII Ed., 8301).U.S. Pat. No. 7,045,620 B1 discloses polymorphic forms of rifaximin. Theapplications and patents referred to here are incorporated herein byreference in their entirety for all purposes

A rifamycin class antibiotic is, for example, a compound having thestructure of Formula I:

wherein A may be the structure A₁:

-   -   or the structure A₂

wherein, -x- is a covalent chemical bond or nil; R is hydrogen oracetyl;

R₁ and R₂ independently represent hydrogen, (C₁₋₄) alkyl, benzyloxy,mono-and di-(C₁₋₃) alkylamino-(C₁₋₄) alkyl, (C₁₋₃)alkoxy-(C₁₋₄)alkyl,hydroxymethyl, hydroxy-(C₂₋₄)-alkyl, nitro or R₁ and R₂ taken togetherwith two consecutive carbon atoms of the pyridine nucleus form a benzenering unsubstituted or substituted by one or two methyl or ethyl groups;R₃ is a hydrogen atom or nil; with the proviso that, when A is A₁, -x-is nil and R₃ is a hydrogen atom; with the further proviso that, when Ais A₂, -x- is a covalent chemical bond and R₃ is nil.

Also described herein is a compound as defined above, wherein A is A₁ orA₂ as above indicated, -x- is a covalent chemical bond or nil, R ishydrogen or acetyl, R₁ and R₂ independently represent hydrogen,(C₁₋₄)alkyl, benzyloxy, hydroxy-(C₂₋₄) alkyl, di-(C₁₋₃)alkylamino-(C₁₋₄) alkyl, nitro or R₁ and R₂ taken together with twoconsecutive carbon atoms of the pyridine nucleus form a benzene ring andR₃ is a hydrogen atom or nil; with the proviso that, when A is A₁, -x-is nil and R₃ is a hydrogen atom; with the further proviso that, when Ais A₂, -x- is a covalent chemical bond and R₃ is nil.

Also described herein is a compound as defined above, wherein A is A₁ orA₂ as above indicated, -x- is a covalent chemical bond or nil, R isacetyl, R₁ and R₂ independently represent hydrogen, (C₁₋₄) alkyl or R₁and R₂ taken together with two consecutive carbon atoms of the pyridinenucleus form a benzene ring and R₃ is a hydrogen atom or nil; with theproviso that, when A is A₁, -x- is nil and R₃ is a hydrogen atom; withthe further proviso that, when A is A₂, -x- is a covalent chemical bondand R₃ is nil.

Also described herein is a compound as defined above, which is4-deoxy-4′-methyl-pyrido[1′,2′-1,2]imidazo [5,4-c]rifamycin SV. Alsodescribed herein is a compound as defined above, which is 4-deoxy-pyrido[1′,2′:1,2]imidazo [5,4-c] rifamycin SV.

Also described herein is a compound as defined above, wherein A is asdescribed above,-x- is a covalent chemical bond or nil; R is hydrogen oracetyl; R₁ and R₂ independently represent hydrogen, (C₁₋₄) alkyl,benzyloxy, mono- and di-(C₁₋₃)alkylamino(C₁₋₄)alkyl,(C₁₋₃)alkoxy-(C₁₋₄)alkyl, hydroxymethyl, hydroxy-(C₂₋₄)-alkyl, nitro orR₁ and R₂ taken together with two consecutive carbon atoms of thepyridine nucleus form a benzene ring unsubstituted or substituted by oneor two methyl or ethyl groups; R₃ is a hydrogen atom or nil; with theproviso that, when A is A₁, -x- is nil and R₃ is a hydrogen atom; withthe further proviso that, when A is A₂, -x- is a covalent chemical bondand R₃ is nil.

Rifaximin is a compound having the structure of formula II:

In certain embodiments, the antibiotic comprises one or more of arifamycin, aminoglycoside, amphenicol, ansamycin, β-Lactam, carbapenem,cephalosporin, cephamycin, monobactam, oxacephem, lincosamide,macrolide, polypeptide, tetracycline, or a 2,4-diaminopyrimidine classantibiotic. Exemplary antibiotics of these classes are listed below.

Rifaximin exerts a broad antibacterial activity in the gastrointestinaltract against localized gastrointestinal bacteria that cause infectiousdiarrhea, including anaerobic strains. It has been reported thatrifaximin is characterized by a negligible systemic absorption, due toits chemical and physical characteristics (Descombe J. J. et al.Pharmacokinetic study of rifaximin after oral administration in healthyvolunteers. Int J Clin Pharmacol Res, 14 (2), 51-56, (1994)).

Without wishing to be bound by any particular scientific theories,rifaximin acts by binding to the beta-subunit of the bacterialdeoxyribonucleic acid-dependent ribonucleic acid (RNA) polymerase,resulting in inhibition of bacterial RNA synthesis. It is active againstnumerous gram (+) and (−) bacteria, both aerobic and anaerobic. In vitrodata indicate rifaximin is active against species of Staphylococcus,Streptococcus, Enterococcus, and Enterobacteriaceae. Bacterial reductionor an increase in antimicrobial resistance in the colonic flora does notfrequently occur and does not have a clinical importance. Rifaximin iscurrently approved in 17 countries outside the US and was licensed bythe Food and Drug Administration (FDA) for the US in May 2004.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention as claimed. In thisapplication, the use of the singular includes the plural unlessspecifically stated otherwise. In this application, the use of “or”means “and/or” unless stated otherwise. Furthermore, the use of the term“including”, as well as other forms, such as “includes” and “included”,is not limiting. Also, terms such as “element” or “component” encompassboth elements and components comprising one unit and elements andcomponents that comprise more than one subunit unless specificallystated otherwise. Also, the use of the term “portion” can include partof a moiety or the entire moiety.

All documents, or portions of documents, cited in this application,including but not limited to patents, patent applications, articles,books, and treatises, are hereby expressly incorporated by reference intheir entirety for any purpose.

One embodiment is a method of treating or preventing hepaticencephalopathy (HE) by administering a therapeutically effective amountof a gastrointestinal (GI) specific antibiotic to a subject. Examples ofgastrointestinal antibiotics as used herein include rifamycin classantibiotics, such as rifaximin.

Embodiments of the invention relate to the discovery of the efficacy ofgastrointestinal (GI) specific antibiotics for the treatment andprevention of Hepatic Encephalopathy. Embodiments relate to the use ofGI specific antibiotics to prevent the onset of HE symptoms and also tolengthen the time to a first breakthrough HE episode. In one embodiment,the time to a first breakthrough HE episode was measured by an increaseof the Conn score to Grade≥2 (e.g., 0 or 1 to ≥2) or a Conn andasterixis score increase of one grade each for those subjects that havea baseline Conn Score of 0. In another embodiment, the time tobreakthrough HE episode was measured by the time to any increase frombaseline in either the Conn score (mental state grade) or asterixisgrade, with Kaplan-Meier estimates of cumulative proportions of subjectswith any increase at Days 28, 56, 84, 112, 140, and 168.

Another embodiment was a measurement of the time to a first HE-relatedhospitalization or the time to development of spontaneous bacterialperitonitis (SBP). Another embodiment was a mean change from baseline inblood ammonia concentration over time or a mean change from baseline incritical flicker frequency values over time. An additional embodimentwas indicated by a mean daily lactulose consumption over time, shiftsfrom baseline in Conn scores over time; or shifts from baseline inasterixis grades over time. Unless otherwise specified, a shift of avalue is the change of that value from a baseline value.

Other measures of efficacy of the treatments described herein includedmean change from baseline in Chronic Liver Disease Questionnaire (CLDQ)scores over time; mean change from baseline in Epworth Sleepiness Scalescores over time; and proportion of subjects who have an EpworthSleepiness Scale score>10. The evaluation of severity of persistenthepatic encephalopathy may also be based, for example, on Conn scores.

In another embodiment, a subject suffering from, susceptible to or inremission from hepatic encephalopathy (HE) can be administered arifamycin class antibiotic for between about 24 weeks and 24 months. Intreating HE, the rifamycin class antibiotic may be administered to thesubject for 12 months and longer, for example for a subject's entirelife span. In one embodiment, the antibiotic is administered daily untilthe death of the subject.

In one embodiment, the invention relates to a method of decreasing asubject's risk of having a breakthrough event by administering to thesubject a GI specific antibiotic. In one embodiment, the for subjectshaving a last HE episode equal to or greater than 90 days prior tostarting on treatment, the risk of failure occurrence was reduced by58%. In another embodiment, the risk of failure occurrence was reducedby between about 30-70%. In another embodiment, the risk was reduced byabout 40% to 70%. In one embodiment the invention relates to decreasingthe risk for episodes of overt hepatic encephalopathy in patientssuffering from HE. In one embodiment, the patients are over 18 years ofage.

In one embodiment, for subjects having a last HE episode more than 90days prior to administration of a GI specific antibiotic, the risk offailure occurrence was decreased by between about 60%. In anotherembodiment, the risk of failure occurrence was decreased by betweenabout 2%-80%.

In another embodiment, for subjects having two or fewer HE episodes inthe six months prior to starting on treatment, the risk of abreakthrough HE episode was decreased by about a 56%. In one embodiment,the risk of a breakthrough HE episode was decreased by between about a20%-70%.

In another embodiment, for subjects having greater than two HE episodesin the six months prior to starting on treatment, the risk of abreakthrough HE episode was reduced by about 63%. In another embodiment,the risk was reduced by about 30%-80%.

In one embodiment, the therapeutically effective amount of agastrointestinal (GI) specific antibiotic comprises from between about1000 mg to about 1200 mg/day.

In one embodiment, the therapeutically effective amount of agastrointestinal (GI) specific antibiotic comprises from between about1100 mg to about 1200 mg/day.

According to one embodiment, the therapeutically effective amount of agastrointestinal (GI) specific antibiotic comprises about 1150 mg/day.

In another embodiment, the therapeutically effective amount is a dosageregimen of one capsule or tablet of the formulation two times each day,wherein each tablet comprises about 550 mg of the gastrointestinal (GI)specific antibiotic, such as rifaximin.

In one embodiment, the therapeutically effective amount is a dosageregimen of two capsules or tablets three times each day, wherein eachcapsule comprises about 200 mg of the gastrointestinal (GI) specificantibiotic.

In one embodiment, the therapeutically effective amount is a dosage of275 mg of a gastrointestinal (GI) specific antibiotic administered fourtimes per day. In another embodiment, 275 mg of a gastrointestinal (GI)specific antibiotic is administered as two dosage forms two times perday.

Another embodiment is a method of maintaining remission of HE in asubject by administering a GI specific antibiotic to the subject.

Another embodiment is a method of increasing time to hospitalization fortreatment of HE by administering to the subject a GI specificantibiotic. In one embodiment, the administration of a GI specificantibiotic reduces hospitalization frequency by about 48%. In anotherembodiment, a GI specific antibiotic reduces hospitalization frequencyby from between about 13% to about 69%.

In one embodiment, treatment with the gastrointestinal (GI) specificantibiotic maintains remission of HE in the subject.

In one embodiment, the GI specific antibiotic is administered to thesubject for six months, one year, two to three years or daily until thesubject's death.

In one embodiment, a Conn score for the subject is improved overbaseline following administration of a GI specific antibiotic.

In one embodiment, a quality of life (QoL) measurement is improved frombaseline with administration of a GI specific antibiotic over a courseof treatment with rifaximin. In one embodiment, the improvised qualityis an improvement in the AUC or TWA of the Chronic Liver DiseaseQuestionnaire (CLDQ).

In one embodiment, the GI specific antibiotic is administered to thesubject with lactulose, prior to treatment with lactulose, or followingtreatment with lactulose. In one embodiment the subject or a health careworker is advised to administer the GI specific antibiotic withlactulose. In one embodiment the subject or a health care worker isadvised by a pharmaceutical label or insert to administer the GIspecific antibiotic with lactulose in order to maintain remission of HE,or to decrease the risk for episodes of overt HE. In one embodiment, thesubject or health care worker is advised to administer two 550 mgtablets of rifaximin twice daily with lactulose. Lactulose use may betitrated over time so that the subject maintains 2-3 soft stool bowelmovements per day. In one embodiment the lactulose is administered in 15ml dosages, wherein each 15 ml dosage contains 10 mg of lactulose. In atypical titration, the subject may start on one dosage, or a partialdosage, per day and then move up in 15 ml dosages over time until theyreach an end point of 2-3 soft stool bowel movements per day.

In one embodiment, subjects in need of treatment for HE and having aChild-Pugh grade of A or B are treated with a GI specific antibiotic. Inanother embodiment, subjects in need of treatment for HE having aChild-Pugh grade of A or B are treated with a GI specific antibiotic incombination with lactulose. In another embodiment, subjects having aChild-Pugh grade of A or B, or their health care worker, are advisedthat they should be treated with a GI specific antibiotic. The advicecan be oral or written advice, such as on a pharmaceutical label orpackage insert. In another embodiment, subjects having a Child-Pughgrade of A or B, or their health care worker, are advised that theyshould be treated with a GI specific antibiotic in combination withlactulose. In one embodiment, a subject in need of treatment for HE andhaving a Child-Pugh grade of less than C is treated with a GI specificantibiotic. In one embodiment, a subject in need of treatment for HE andhaving a Child-Pugh grade of less than C is treated with a GI specificantibiotic and lactulose.

In another embodiment, a subject in need of treatment for HE, or theirhealth care worker is advised of the risk for anaphylaxis prior totreatment with a GI specific antibiotic.

In one embodiment, the GI specific antibiotic is administered with oneor more of align, alinia, Lactulose, pentasa, cholestyramine,sandostatin, vancomycin, lactose, amitiza, flagyl, zegerid, prevacid, ormiralax.

In one embodiment, following treatment with GI specific antibiotic, aConn score (mental state grade) of a subject decreases.

In one embodiment, following treatment with a GI specific antibiotic, aConn score increase from baseline is increased.

In one embodiment, following treatment with a GI specific antibiotic, adelay in time to an increase in Conn score is about 54%. For example,the percentage delay in time to increase in Conn score may be betweenabout 30% to about 70%.

In another embodiment, administration of the GI specific antibioticprevents an increase in Conn score. For example, administration of theGI specific antibiotic increases the time to an increase from baselinein a Conn score.

In one embodiment, administration of the GI specific antibiotic resultsin an increase of time to an increase from baseline in an asterixisgrade.

In another embodiment, administration of the GI specific antibioticresults in a delay in the time to increase in asterixis grade.

In another embodiment, administration of the GI specific antibioticresults in an increase in time to first HE-related hospitalization.

In another embodiment, administration of the GI specific antibioticresults in an increase in the time to development of spontaneousbacterial peritonitis (SBP).

In another embodiment, administration of the GI specific antibioticresults in a decrease in blood ammonia concentration from baseline afteradministration of rifaximin. For example, the decrease in blood ammoniaconcentration may be from baseline to 170 days of about 6 μg/dL.

In another embodiment, administration of the GI specific antibioticresults in an increase in critical flicker frequency values frombaseline after administration of rifaximin.

In another embodiment, administration of the GI specific antibioticresults in a decrease in daily lactulose consumption from baseline overtime after administration with rifaximin.

In another embodiment, administration of the GI specific antibioticresults in a decrease in daily lactulose consumption is from betweenabout 7 doses of lactulose to about 2 doses of lactulose.

In another embodiment, administration of the GI specific antibioticresults in a lactulose use that initially increases from baseline. Forexample, the lactulose use may be from between about 1 and about 30days.

In another embodiment, administration of the GI specific antibioticresults in a shift in baseline in Conn scores over time afteradministration of rifaximin. For example, the shift in baseline in Connscores may be from between about 1 to about 2.

In another embodiment, administration of the GI specific antibioticresults in a shift from baseline in asterixis grades over time.

In another embodiment, administration of the GI specific antibioticresults in a change from baseline in Chronic Liver Disease Questionnaire(CLDQ) scores over time.

In another embodiment, administration of the GI specific antibioticresults in a change from baseline in Epworth Sleepiness Scale scoresover time after administration of rifaximin.

As is known, the Model for End-Stage Liver Disease (MELD) score can beutilized to predict liver disease severity based on serum creatinine,serum total bilirubin, and the international normalized ratio forprothrombin time INR. The MELD score and has been shown to be useful inpredicting mortality in patients with compensated and decompensatedcirrhosis. The maximum score given for MELD is 40. All values higherthan 40 are given a score of 40.

In another embodiment, subjects having a MELD level of between about 1to 24 responded to treatment for HE using administration of the GIspecific. In another embodiment, subjects having a MELD level less thanor equal to 10 responded to treatment with GI specific antibiotics. Inanother embodiment, subjects having a MELD level between 11 and 18respond to treatment with GI specific antibiotics. In anotherembodiment, subjects having a MELD level between 19 and 24 respond totreatment with GI specific antibiotics. In one embodiment, subjects inneed of treatment for HE and having a MELD score of 25 or less aretreated with a GI specific antibiotic. In another embodiment, subjectsin need of treatment for HE having a MELD score of 25 or less aretreated with a GI specific antibiotic in combination with lactulose. Inanother embodiment, subjects having a MELD score of 25 or less areadvised that they should be treated with a GI specific antibiotic. Theadvice can be oral or written advise, such as on a pharmaceutical labelor package insert. In another embodiment, subjects having a MELD scoreof 25 or less are advised that they should be treated with a GI specificantibiotic in combination with lactulose.

One embodiment of the invention is a method of treating or preventing HEby administering 1100 mg of rifaximin per day to a patient for more than28 days.

Another embodiment is a method of decreasing lactulose use in a subject.This method includes: administering rifaximin to a subject daily that isbeing treated with lactulose, and tapering lactulose consumption. Forexample, the lactulose consumption may be reduced by 1, 2, 3, 4, 5, 6 ormore unit dose cups of lactulose from a baseline level. Alternatively,the lactulose use may be reduced by 5, 10, 15, 20, 25, 30, 34, 40, 45,50, 55, 60, 65, or 70 g lactulose from a baseline level. In oneembodiment, the baseline use of lactulose is no use.

One embodiment of the invention is a method of maintaining remission ofHE in a subject comprising administering 550 mg of rifaximin twice a day(BID) to the subject.

Another embodiment is a method of increasing time to hospitalization fortreatment of HE comprising, administering to a subject 550 mg ofrifaximin two times per day (BID).

The term “administration” or “administering” includes routes ofintroducing a GI specific antibiotic to a subject to perform theirintended function. Examples of routes of administration that may be usedinclude injection (subcutaneous, intravenous, parenterally,intraperitoneally, intrathecal), oral, inhalation, rectal andtransdermal. The pharmaceutical preparations may be given by formssuitable for each administration route. For example, these preparationsare administered in tablets or capsule form, by injection, inhalation,eye lotion, eye drops, ointment, suppository, etc. administration byinjection, infusion or inhalation; topical by lotion or ointment; andrectal by suppositories. Oral administration is preferred. The injectioncan be bolus or can be continuous infusion. Depending on the route ofadministration, a GI specific antibiotic can be coated with or disposedin a selected material to protect it from natural conditions that maydetrimentally effect its ability to perform its intended function. A GIspecific antibiotic can be administered alone, or in conjunction witheither another agent or agents as described above or with apharmaceutically-acceptable carrier, or both. A GI specific antibioticcan be administered prior to the administration of the other agent,simultaneously with the agent, or after the administration of the agent.Furthermore, a GI specific antibiotic can also be administered in aproform, which is converted into its active metabolite, or more activemetabolite in vivo.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

As will be readily apparent to one skilled in the art, the useful invivo dosage to be administered and the particular mode of administrationwill vary depending upon the age, weight and mammalian species treated,the particular compounds employed, and the specific use for which thesecompounds are employed. The determination of effective dosage levels,that is the dosage levels necessary to achieve the desired result, canbe accomplished by one skilled in the art using routine pharmacologicalmethods. Typically, human clinical applications of products arecommenced at lower dosage levels, with dosage level being increaseduntil the desired effect is achieved.

As used herein, an “increase” or “decrease” in a measurement, unlessotherwise specified, is typically in comparison to a baseline value. Forexample, an increase in time to hospitalization for subjects undergoingtreatment may be in comparison to a baseline value of time tohospitalization for subjects that are not undergoing such treatment. Insome instances an increase or decrease in a measurement can be evaluatedbased on the context in which the term is used.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers which are nontoxic to the cell or mammalbeing exposed thereto at the dosages and concentrations employed. Oftenthe physiologically acceptable carrier is an aqueous pH bufferedsolution. Examples of physiologically acceptable carriers includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN, polyethylene glycol (PEG).

The term “effective amount” includes an amount effective, at dosages andfor periods of time necessary, to achieve the desired result, e.g.,sufficient to treat or prevent HE in a patient or subject. An effectiveamount of a GI specific antibiotic may vary according to factors such asthe disease state, age, and weight of the subject, and the ability of aGI specific antibiotic to elicit a desired response in the subject.Dosage regimens may be adjusted to provide the optimum therapeuticresponse. An effective amount is also one in which any toxic ordetrimental effects (e.g., side effects) of a GI specific antibiotic areoutweighed by the therapeutically beneficial effects.

“Ameliorate,” “amelioration,” “improvement” or the like refers to, forexample, a detectable improvement or a detectable change consistent withimprovement that occurs in a subject or in at least a minority ofsubjects, e.g., in at least about 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%,50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 100% or in a range betweenabout any two of these values. Such improvement or change may beobserved in treated subjects as compared to subjects not treated withrifaximin, where the untreated subjects have, or are subject todeveloping, the same or similar disease, condition, symptom or the like.Amelioration of a disease, condition, symptom or assay parameter may bedetermined subjectively or objectively, e.g., self assessment by asubject(s), by a clinician's assessment or by conducting an appropriateassay or measurement, including, e.g., a quality of life assessment suchas a Chronic Liver Disease Questionnaire (CLDQ), a slowed progression ofa disease(s) or condition(s), a reduced severity of a disease(s) orcondition(s), or a suitable assay(s) for the level or activity(ies) of abiomolecule(s), cell(s) or by detection of HE episodes in a subject.Amelioration may be transient, prolonged or permanent or it may bevariable at relevant times during or after a GI specific antibiotic isadministered to a subject or is used in an assay or other methoddescribed herein or a cited reference, e.g., within timeframes describedinfra, or about 1 hour after the administration or use of a GI specificantibiotic to about 28 days, or 1, 3, 6, 9 months or more after asubject(s) has received such treatment.

The “modulation” of, e.g., a symptom, level or biological activity of amolecule, or the like, refers, for example, that the symptom oractivity, or the like is detectably increased or decreased. Suchincrease or decrease may be observed in treated subjects as compared tosubjects not treated with a GI specific antibiotic, where the untreatedsubjects have, or are subject to developing, the same or similardisease, condition, symptom or the like. Such increases or decreases maybe at least about 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%,75%, 80%, 85%, 90%, 95%, 98%, 100%, 150%, 200%, 250%, 300%, 400%, 500%,1000% or more or within any range between any two of these values.Modulation may be determined subjectively or objectively, e.g., by thesubject's self assessment, by a clinician's assessment or by conductingan appropriate assay or measurement, including, e.g., quality of lifeassessments or suitable assays for the level or activity of molecules,cells or cell migration within a subject. Modulation may be transient,prolonged or permanent or it may be variable at relevant times during orafter a GI specific antibiotic is administered to a subject or is usedin an assay or other method described herein or a cited reference, e.g.,within times descried infra, or about 1 hour of the administration oruse of a GI specific antibiotic to about 3, 6, 9 months or more after asubject(s) has received a GI specific antibiotic.

The term “modulate” may also refer to increases or decreases in theactivity of a cell in response to exposure to a GI specific antibiotic,e.g., the inhibition of proliferation and/or induction ofdifferentiation of at least a sub-population of cells in an animal suchthat a desired end result is achieved, e.g., a therapeutic result of GIspecific antibiotic used for treatment may increase or decrease over thecourse of a particular treatment.

The term “obtaining” as in “obtaining a GI specific antibiotic” isintended to include purchasing, synthesizing or otherwise acquiring a GIspecific antibiotic.

The phrases “parenteral administration” and “administered parenterally”as used herein includes, for example, modes of administration other thanenteral and topical administration, usually by injection, and includes,without limitation, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticulare, subcapsular, subarachnoid, intraspinal and intrasternalinjection and infusion.

The language “a prophylactically effective amount” of a compound refersto an amount of a GI specific antibiotic which is effective, upon singleor multiple dose administration to the subject, in preventing ortreating HE.

Pharmaceutical Preparations

Embodiments also provide pharmaceutical compositions, comprising aneffective amount of a rifaximin described herein and a pharmaceuticallyacceptable carrier. In a further embodiment, the effective amount iseffective to treat a bacterial infection, Crohn' s disease, hepaticencephalopathy, antibiotic associated colitis, and/or diverticulardisease in a subject further suffering from hepatic insufficiency.

Embodiments also provide pharmaceutical compositions comprisingrifaximin and a pharmaceutically acceptable carrier. Doses may beselected, for example on the basis of desired amounts of systemicadsorption, elimination half-life, serum concentration and the like.Embodiments of the pharmaceutical composition further compriseexcipients, for example, one or more of a diluting agent, binding agent,lubricating agent, disintegrating agent, coloring agent, flavoring agentor sweetening agent. One composition may be formulated for selectedcoated and uncoated tablets, hard and soft gelatin capsules,sugar-coated pills, lozenges, wafer sheets, pellets and powders insealed packet. For example, compositions may be formulated for topicaluse, for example, ointments, pomades, creams, gels and lotions.

In an embodiment, rifaximin is administered to the subject using apharmaceutically-acceptable formulation, e.g., apharmaceutically-acceptable formulation that provides sustained deliveryof the rifaximin to a subject for at least 12 hours, 24 hours, 36 hours,48 hours, one week, two weeks, three weeks, or four weeks after thepharmaceutically-acceptable formulation is administered to the subject.

In certain embodiments, these pharmaceutical compositions are suitablefor topical or oral administration to a subject. In other embodiments,as described in detail below, the pharmaceutical compositions of thepresent invention may be specially formulated for administration insolid or liquid form, including those adapted for the following: (1)oral administration, for example, drenches (aqueous or non-aqueoussolutions or suspensions), tablets, boluses, powders, granules, pastes;(2) parenteral administration, for example, by subcutaneous,intramuscular or intravenous injection as, for example, a sterilesolution or suspension; (3) topical application, for example, as acream, ointment or spray applied to the skin; (4) intravaginally orintrarectally, for example, as a pessary, cream or foam; or (5) aerosol,for example, as an aqueous aerosol, liposomal preparation or solidparticles containing the compound.

The phrase “pharmaceutically acceptable” refers to rifaximincompositions containing rifaximin and/or dosage forms which are, withinthe scope of sound medical judgment, suitable for use in contact withthe tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” includespharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject chemical fromone organ, or portion of the body, to another organ, or portion of thebody. Each carrier is preferably “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the subject. Some examples of materials which can serve aspharmaceutically-acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21)other non-toxic compatible substances employed in pharmaceuticalformulations.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically-acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Compositions containing a rifaximin forms disclosed herein include thosesuitable for oral, nasal, topical (including buccal and sublingual),rectal, vaginal, aerosol and/or parenteral administration. Thecompositions may conveniently be presented in unit dosage form and maybe prepared by any methods well known in the art of pharmacy. The amountof active ingredient which can be combined with a carrier material toproduce a single dosage form will vary depending upon the host beingtreated, the particular mode of administration. The amount of activeingredient which can be combined with a carrier material to produce asingle dosage form will generally be that amount of the compound whichproduces a therapeutic effect. Generally, out of one hundred %, thisamount will range from about 1% to about ninety-nine % of activeingredient, preferably from about 5% to about 70%, most preferably fromabout 10% to about 30%.

Methods of preparing these compositions include the step of bringinginto association a rifaximin with the carrier and, optionally, one ormore accessory ingredients. In general, the formulations are prepared byuniformly and intimately bringing into association a rifaximin withliquid carriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product.

Compositions suitable for oral administration may be in the form ofcapsules, cachets, pills, tablets, lozenges (using a flavored basis,usually sucrose and acacia or tragacanth), powders, granules, or as asolution or a suspension in an aqueous or non-aqueous liquid, or as anoil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup,or as pastilles (using an inert base, such as gelatin and glycerin, orsucrose and acacia) and/or as mouth washes and the like, each containinga predetermined amount of a rifaximin as an active ingredient. Acompound may also be administered as a bolus, electuary or paste.

The term “pharmaceutical agent composition” (or agent or drug) as usedherein refers to a chemical compound, composition, agent or drug capableof inducing a desired therapeutic effect when properly administered to apatient. It does not necessarily require more than one type ofingredient.

The compositions may be in the form of tablets, capsules, powders,granules, lozenges, liquid or gel preparations. Tablets and capsules fororal administration may be in a form suitable for unit dose presentationand may contain conventional excipients. Examples of these are: bindingagents such as syrup, acacia, gelatin, sorbitol, tragacanth, andpolyvinylpyrrolidone; fillers such as lactose, sugar, maize-starch,calcium phosphate, sorbitol or glycine; tableting lubricants, such asmagnesium stearate, silicon dioxide, talc, polyethylene glycol orsilica; disintegrants, such as potato starch; or acceptable wettingagents, such as sodium lauryl sulfate. The tablets may be coatedaccording to methods well known in normal pharmaceutical practice. Oralliquid preparations may be in the form of, for example, aqueous or oilysuspensions, solutions, emulsions, syrups or elixirs, or may bepresented as a dry product for reconstitution with water or othersuitable vehicle before use. Such liquid preparations may containconventional additives such as suspending agents, e.g., sorbitol, syrup,methyl cellulose, glucose syrup, gelatin, hydrogenated edible fats,emulsifying agents, e.g., lecithin, sorbitan monooleate, or acacia;non-aqueous vehicles (including edible oils), e.g., almond oil,fractionated coconut oil, oily esters such as glycerine, propyleneglycol, or ethyl alcohol; preservatives such as methyl or propylp-hydroxybenzoate or sorbic acid, and, if desired, conventionalflavoring or coloring agents.

The phrases “systemic administration,” “administered systemically,”“peripheral administration,” and “administered peripherally,” as usedherein mean the administration of a GI specific antibiotic, drug orother material, such that it enters the subject's system and, thus, issubject to metabolism and other like processes, for example,subcutaneous administration.

The language “therapeutically effective amount” of a GI specificantibiotic refers to an amount of a GI specific antibiotic which iseffective, upon single or multiple dose administration to the subject,in inhibiting the bacterial growth and/or invasion, or in decreasingsymptoms, such as HE episodes, relating to bacterial growth in asubject. “Therapeutically effective amount” also refers to the amount ofa therapy (e.g., a composition comprising a GI specific antibiotic),which is sufficient to reduce the severity of HE in a subject.

As used herein, the terms “prevent,” “preventing,” and “prevention”refer to the prevention of the recurrence, onset, or development HEepisodes or more symptoms of HE. Preventing includes protecting againstthe occurrence and severity of HE episodes.

As used herein, the term “prophylactically effective amount” refers tothe amount of a therapy (e.g., a composition comprising a GI specificantibiotic) which is sufficient to result in the prevention of thedevelopment, recurrence, or onset of HE episodes or to enhance orimprove the prophylactic effect(s) of another therapy.

“Rifaximin”, as used herein, includes solvates and polymorphous forms ofthe molecule, including, for example, α, β, γ, δ, ε, η, ζ and amorphousforms of rifaximin. These forms are described in more detail, forexample, in U.S. Ser. No. 11/873,841; U.S. Ser. No. 11/658,702; EP 05004 635.2, filed 3 May 2005; U.S. Pat. No. 7,045,620; U.S. 61/031,329;and G. C. Viscomi, et al., Cryst Eng Comm, 2008, 10, 1074-1081 (April2008). Each of these references is hereby incorporated by reference inentirety.

The forms of rifaximin can be advantageously used in the production ofmedicinal preparations having antibiotic activity, containing rifaximin,for both oral and topical use. The medicinal preparations for oral usemay contain rifaximin α or β or γ together with other excipients, forexample diluting agents such as mannitol, lactose and sorbitol; bindingagents such as starchs, gelatines, sugars, cellulose derivatives,natural gums and polyvinylpyrrolidone; lubricating agents such as talc,stearates, hydrogenated vegetable oils, polyethylenglycol and colloidalsilicon dioxide; disintegrating agents such as starchs, celluloses,alginates, gums and reticulated polymers; coloring, flavoring andsweetening agents.

Medicinal preparations may contain gastrointestinal specific antibioticstogether with usual excipients, such as white petrolatum, white wax,lanoline and derivatives thereof, stearylic alcohol, red iron oxide,propylene glycol, talc, sodium lauryl sulfate, ethers of fattypolyoxyethylene alcohols, disodium edentate, glycerol palmitostearate,esters of fatty polyoxyethylene acids, sorbitan monostearate, glycerylmonostearate, propylene glycol monostearate, hypromellose, polyethyleneglycols, sodium starch glycolate, methylcellulose, hydroxymethylpropylcellulose, sodium carboxymethylcellulose, microcrystallinecellulose, colloidal aluminium and magnesium silicate, titanium dioxide,propylene glycol, colloidal silicon dioxide, or sodium alginate.

As used herein, “breakthrough HE,” includes, for example, an increase ofthe Conn score to Grade≥2 (e.g., 0 or 1 to ≥2) or a Conn and Asterixisscore increase of 1 grade each for those subjects that have a baselineConn score of 0.

As used herein, “time to the first breakthrough HE episode,” includes,for example, the duration between the date of first administration ofrifaximin and the date of first breakthrough HE episode.

As used herein, the term “breakthrough HE event”, is intended to includea marked, clinically significant deterioration in neurological functioncaused by toxic substances accumulating in the blood that cause adeleterious effect on self care, and often leads to hospitalization.Breakthrough HE event is also defined as an increase of a Conn Scoreto >2 (i.e., 0 or 1 to ≥2) or a Conn score and asterixis grade increaseof 1 each for those subjects that have a baseline Conn score of 0.

Provided herein are methods for determining if a subject has aneurological condition by determining the CFF of a subject at two ormore time points. In exemplary embodiments, time points can be 1, 2, 3,4, 5, 6 or 7 days apart; or 2, 3, or 4 weeks apart; or 2, 3, 4, 5, 6, 7,8, 9, 10, 11 or 12 months apart or any time point in between any twovalues. In other embodiments, a subject may be monitored at routineintervals for life.

The methods of the invention provide that a decrease in CFF between twoor more time points is indicative that the probability of an HEbreakthrough event is approaching. Moreover, if a subject has a CFF twavalue at a time point that is less than 24 Hz, it is indicative that thesubject has an increased probability of an HE event. Therefore, adecrease between CFF in two or more time points or a twa of 24 Hz orless is indicative that the subject has HE, has an increased chance ofan HE breakthrough event, and/or should be treated with Rifaximin.Accordingly, based on the data collected to date, in one embodimentprovided herein are methods of determining if a subject has HE, ofpredicting the occurrence of a breakthrough HE event, or determining theprognosis of a subject by determining a subject's CFF is below 24 Hz,wherein a CFF below 24 Hz is indicative that the subject has HE, islikely to have a breakthrough HE event, or has a poor prognosis. Incertain embodiments, a CFF of less than 24 Hz is indicative that a GIspecific antibiotic, e.g., rifaximin, should be administered.

Provided herein are prognostic methods based on determining the CFF ortwa CFF wherein a twa CFF of less than 24 is indicative of poorprognosis, or wherein a decrease in CFF or twa CFF between measurementsat different time points is indicative of poor prognosis. Poor prognosisincludes the survival of the subject for less than 2, 3, 4, 5, 6, 7, 8or more years or as described herein or in the opinion of a healthcareprofessional, the subject or a person observing the subject.

In other embodiments, provided herein are method for determining if asubject has HE or has an increased risk of having a HE breakthroughevent by measuring the venous ammonia level in a subject at two or moretime points, wherein an increase in the venous ammonia level isindicative that the subject has HE, has an increased chance of an HEbreakthrough event, and/or should be treated with a GI specificantibiotic, e.g., rifaximin. In certain embodiments the venous ammonialevel is a time weighted average venous ammonia level.

Venous ammonia concentration can be measured using methods that areknown to one of skill in the art. The accuracy of ammonia determinationis dependent on sample collection. Whole blood is preferred. In onespecific method described herein, blood is collect blood from astasis-free vein into an EDTA evacuated tube. The sample is placed inice immediately after collecting and mixing. The sample is placed in acold environment, e.g., on ice, for approximately ten minutes and thencentrifuged. The plasma is separated from the sample within fifteenminutes of collection and frozen. Hemolyzed samples should not be usedfor further analysis.

The frozen sample is subjected to an enzymatic assay to determine theamount of ammonia present in the sample. The sample containing ammoniais mixed with α-ketoglutarate and reduced nicotinamide adeninedinucleotide phosphate (NADPH) to form L-glutamate and NADP and water.The reaction is catalyzed by glutamate dehydrogenase. The results aredetermined spectrophotometrically by monitoring the decrease inabsorbance at 340 nm due to the oxidation of NADPH. This decrease isproportional to the ammonia concentration.

In other embodiments, provided herein are methods for determining if asubject has a neurological condition by measuring the CFF between two ormore time points. A decrease in the CFF between time points isindicative that a subject has a neurological condition. In certainembodiments, the CFF is the twa of CFF events.

According to one embodiment of the teachings of the present invention,provided herein are a database having a data structure which contains anumber of CFF or venous ammonia levels from subjects. Similarly, atleast one of the databases includes a data structure which maintains anumber of relationships between the CFF or venous ammonia levels and thedisease state of the subjects and that defines the business rules forperforming the methods of the present invention. These business rulescan include defined methods for determining if a subject has HE or is atrisk of having a breakthrough HE event. Likewise, the business rules caninclude defined methods for determining if a subject has a neurologicalcondition. The diagnosis or prognosis can be optionally selected usingthe novel software of the systems and methods of the present invention.In this scenario, the systems and methods, including the novel programconfigurations, will automatically perform the methods of the presentinvention without additional user input.

Provided herein are methods for determining if a subject has aneurological condition by determining the CFF of a subject at two ormore time points.

In particular, one such additional method provided herein includes novelsoftware including a number of program modules or components located ona server within the system for creating and populating a database foruse in the diagnostic or prognostic methods of the invention. In otherwords, in one embodiment, the systems and methods provide for thecreation and management of a particular policy and policy management fora particular client. One of ordinary skill in the art will understandupon reading this disclosure that the various embodiments include novelsoftware including a number of program modules or components located onthe computer based system or network of the present invention, e.g.servers, sending remote clients, and receiving remote clients, forfacilitating the methods of the present invention.

As used herein, “time to first HE-related hospitalization,” includes,for example, the duration between the first dose of rifaximin and thedate of first HE-related hospitalization.

As used herein, “time to an increase from baseline in the Conn score”includes, for example, the duration between the first dose of rifaximinand the date of first increase in Conn score.

As used herein, “time to an increase from baseline in the asterixisgrade”, includes, for example, the duration between the first dose ofrifaximin and the date of first increase in asterixis grade.

As used herein, “mean change from baseline in the fatigue domain scoreof Chronic Liver Disease Questionnaire (CLDQ),at end of treatment (EOT)”is the mean score with a baseline from before the first administrationof rifaximin.

As used herein, “mean change from baseline in blood ammoniaconcentration at EOT,” includes the mean score with a baseline frombefore the first administration of rifaximin.

As used herein, the “time to diagnosis of spontaneous bacterialperitonitis (SBP),” includes, for example, the duration between thefirst dose of rifaximin and the date of first episode of SBP.

As used herein, the “mean change from baseline at each post-baseline incritical flicker frequency values,” is measured, for example, from abaseline established before the first administration of rifaximin.

“GI specific antibiotic,” and “GI antibiotic” as used herein includeantibiotic known to have an effect on GI disease. For example, arifamycin class antibiotic (e.g., rifaximin), neomycin, metronidazole,teicoplanin, ciprofloxacin, doxycycline, tetracycline, augmentin,cephalexin, penicillin, ampicillin, kanamycin, rifamycin, vancomycin,rifaximin, and combinations thereof are useful GI specific antibiotics.Even more preferable are GI specific antibiotics with low systemicabsorption, for example, rifaximin. Low systemic absorption includes,for example, less than 10% absorption, less than 5% absorption, lessthan 1% absorption and less than 0.5% absorption. Low systemicabsorption also includes, for example, from between about 0.01-1%absorption, from between about 0.05 -1% absorption, from between about0.1-1% absorption, from between about 1-10% absorption, or from betweenabout 5-20% absorption.

As used herein, “subject” includes organisms which are capable ofsuffering from a bowel disorder or other disorder treatable by rifaximinor who could otherwise benefit from the administration of a rifaximin asdescribed herein, such as human and non-human animals. Preferred humananimals include human subjects. The term “non-human animals” of theinvention includes all vertebrates, e.g., mammals, e.g., rodents, e.g.,mice, and non-mammals, such as non-human primates, e.g., sheep, dog,cow, chickens, amphibians, reptiles, etc. Susceptible to a boweldisorder is meant to include subjects at risk of developing a boweldisorder or bowel infection, e.g., subjects suffering from hepaticfailure or decreased hepatic function, immune suppression, subjects thathave been exposed to other subjects with a bacterial infection,physicians, nurses, subjects traveling to remote areas known to harborbacteria that causes travelers' diarrhea, etc.

The language “a prophylactically effective amount” of a compound refersto an amount of a compound of the invention of formula I, formula II, orotherwise described herein which is effective, upon single or multipledose administration to the subject, in preventing or treating hepaticencephalopathy.

Another embodiment includes articles of manufacture that comprise, forexample, a container holding a pharmaceutical composition suitable fororal administration of rifaximin in combination with printed labelinginstructions providing a discussion of when a particular dosage formextends remission of HE or prevents or delays future episodes of HE. Thedosage can be modified for administration to a subject suffering fromHE, or include labeling for administration to a subject suffering fromHE. Exemplary dosage forms and administration protocols are describedinfra. The composition will be contained in any suitable containercapable of holding and dispensing the dosage form and which will notsignificantly interact with the composition and will further be inphysical relation with the appropriate labeling. The labelinginstructions may be consistent with the methods of treatment asdescribed hereinbefore. The labeling may be associated with thecontainer by any means that maintain a physical proximity of the two, byway of non-limiting example, they may both be contained in a packagingmaterial such as a box or plastic shrink wrap or may be associated withthe instructions being bonded to the container such as with glue thatdoes not obscure the labeling instructions or other bonding or holdingmeans.

In one embodiment, the instructions will inform and/or advise a healthcare worker, prescribing physician, a pharmacist, or a subject that theyshould advise a patient suffering from hepatic encephalopathy thatadministration of rifaximin may induce cytochrome P450. In anotherembodiment, the instructions will inform the subject and/or thehealthcare provider that there is an extended time to remission orrelapse of subjects that take rifaximin. In another embodiment, theinstructions will inform the subject and/or the healthcare worker orprovider that rifaximin does not significantly alter the C_(max),AUC_(0-t), or AUC_(0-∞) of midazolam. In another embodiment, theinstructions will inform the subject and/or the healthcare worker orprovider that rifaximin does not increase the risk of QT prolongation.

Packaged compositions are also provided, and may comprise atherapeutically effective amount of rifaximin tablets or capsules. Kitsare also provided herein, for example, kits for treating HE in asubject. The kits may contain, for example, rifaximin and instructionsfor use when treating a subject for an HE. The instructions for use maycontain prescribing information, dosage information, storageinformation, and the like.

Kits may include pharmaceutical preparations of the GI specificantibiotics along with pharmaceutically acceptable solutions, carriersand excipients.

Forms of rifaximin can be advantageously used in the production ofmedicinal preparations having antibiotic activity, containing rifaximin,for both oral and topical use. The medicinal preparations for oral usemay contain one or more forms of rifaximin (for example, α or β or γ)together with other excipients, for example diluting agents such asmannitol, lactose and sorbitol; binding agents such as starchs,gelatines, sugars, cellulose derivatives, natural gums andpolyvinylpyrrolidone; lubricating agents such as talc, stearates,hydrogenated vegetable oils, polyethylenglycol and colloidal silicondioxide; disintegrating agents such as starchs, celluloses, alginates,gums and reticulated polymers; coloring, flavoring and sweeteningagents.

Solid preparations of gastrointestinal specific antibioticsadministrable by the oral route include for instance coated and uncoatedtablets, soft and hard gelatin capsules, sugar-coated pills, lozenges,wafer sheets, pellets and powders in sealed packets.

Medicinal preparations may contain gastrointestinal specific antibioticstogether with usual excipients, such as white petrolatum, white wax,lanoline and derivatives thereof, stearylic alcohol, red iron oxide,propylene glycol, talc, sodium lauryl sulfate, ethers of fattypolyoxyethylene alcohols, disodium edentate, glycerol palmitostearate,esters of fatty polyoxyethylene acids, sorbitan monostearate, glycerylmonostearate, propylene glycol monostearate, hypromellose, polyethyleneglycols, sodium starch glycolate, methylcellulose, hydroxymethylpropylcellulose, sodium carboxymethylcellulose, microcrystallinecellulose, colloidal aluminium and magnesium silicate, titanium dioxide,propylene glycol, colloidal silicon dioxide, or sodium alginate.

West Haven Criteria (Conn Score):

Measurements of change in mental status may be done, for example, by theConn score (also known as the West Haven score). The Conn score has beenwidely used as a measure of mental state in HE studies and is based onthe criteria of Parsons-Smith as modified by Conn. Asterixis will not beconsidered when assessing the subject's status using the Conn scoringcriteria listed below.

The scale used in the Conn scoring system is provided below.

-   -   Grade 0=No personality or behavioral abnormality detected    -   Grade 1=Trivial lack of awareness, euphoria or anxiety;        shortened attention span; impairment of addition or subtraction    -   Grade 2=Lethargy; disorientation for time; obvious personality        change; inappropriate behavior    -   Grade 3=Somnolence to semi-stupor, responsive to stimuli;        confused; gross disorientation; bizarre behavior    -   Grade 4=Coma; unable to test mental state

HE is defined as a spectrum of neuropsychiatric abnormalities seen inpatients with liver dysfunction, diagnosed after routine exclusion ofother known neurologic disease. HE is a major complication of livercirrhosis, affecting 30-45% patients. In 2006, the CDC listed cirrhosisas the 12th leading cause of death by disease in the U.S. HE affects thepatient's consciousness, personality, intellect and neuromuscularfunction, and may range from a minimal disturbance in cognition, tocoma.

In the gut, enteric bacteria act on nitrogen-containing substrates togenerate ammonia. FIG. 15 represents the situation in unaffected HEsubjects: ammonia is removed from the blood as it passes through theliver where it is converted to urea, and excreted by the kidneys. Incirrhosis, ammonia from the intestines bypasses the damaged liver as aresult of vascular shunts. This increases blood ammonia, which passesinto the brain generating glutamine from the amino acid glutamate. Theexcess glutamine causes many deleterious effects on brain function; itinhibits neurotransmission, interferes with mitochondrial energymetabolism, and causes swelling of astrocytes.

The clinical presentation of HE is classified according to the schemeshown in FIG. 16 . HE associated with Cirrhosis—the most common byfar—is type C. HE Type C is sub-classified into episodic, persistent andminimal categories. Episodic and persistent varieties are clinicallyreadily apparent conditions, and hence are denoted as Overt. Episodic HEpresents with impairment in all the neurological functions mentionedabove. As the term episodic implies, there are periods between episodeswhen no distinctive symptoms are seen. Episodes may be precipitated byfactors such as constipation, infection, dehydration, GI hemorrhage andcertain medications. If the cause is not immediately identified, theepisode is referred to as spontaneous.

HE episodes are usually reversible with treatment—but they're oftenrecurring. HE is a clinical diagnosis made by some tools, including theWest Haven, or Conn, Score. In use for about 30 years, The HESA scoringalgorithm (FIG. 17 ) is a relatively new tool used for accurateassignment of Conn criteria. Neuromuscular dysfunction can be measuredby eliciting asterixis, or flapping tremor. Blood ammonia levels areoften measured to support the diagnosis. Neurophysiological tests, suchas critical flicker frequency and EEG, are potentially very useful tosupport the clinical findings. The Conn criteria use an increasing gradeto associate with increasing neurological impairment, (ranging from 0=noimpairment to 4=coma)

-   -   Grades 1, 2, and 3 represent an worsening in impairment in:        -   Consciousness—ranging from a trivial lack of awareness to            somnolence;        -   impairment in intellectual ability and alterations in            personality        -   This assessment can be conducted quickly, requires minimal            intervention from the examiner or cooperation from the            patient,        -   And we often use information from family or caregivers to            help gauge the severity of HE episodes when the patient is            confused.

While patients with grade 1 HE can be managed at home by a caregiver,any escalation to grade 2 or higher may require hospitalization and evenmanagement in intensive care. The Conn criteria use an increasing gradeto associate with increasing neurological impairment, (ranging from 0=noimpairment to 4=coma). Grades 1, 2, and 3 represent an worsening inimpairment in: consciousness; intellectual ability and alterations inpersonality. This assessment can be conducted quickly, requires minimalintervention from the examiner or cooperation from the patient,information from family or caregivers is often used to gauge theseverity of HE episodes.

While patients with grade 1 HE can be managed at home by a caregiver,any escalation to grade 2 or higher may require hospitalization and evenmanagement in intensive care.

There is a similar grading system for asterixis. If an HE patient isasked to hold out their hands just so, a jerky so called asterixis orflapping tremor will be observed. The number of beats is counted andscored from zero for none to four for almost continuous flapping. Thisis a simple test but requires a cooperative and conscious patient.

HE presents a vicious cycle of dysfunction and disability that has adramatic effect on patients, their families and the healthcare system.Early on, impairments in behavior, personality, intellect andconsciousness affect the patient's social and family life and ability tohold employment. As the condition worsens, it impacts capacity for selfcare, medication compliance, lack of compliance further intensifies HEsymptoms and frequency of episodes. As a result, patients may needin-home assistance and often land in the ER or hospital beds. Severe HEcan be a life threatening event, but it more commonly devastates the QOLof patients and their families; some caregivers liken the experience tocaring for unpredictably episodic Alzheimer's disease. Impact oncaregiver is shown in FIG. 18 .

In terms of the impact on healthcare, the number of HE discharges morethan doubled between 1993 to 2007. See FIG. 19 . Costs increased—fromabout 13 k to 30 k per hospitalization. So, the goals for HE Therapyinclude, for example, bringing acute episodes to quick resolution, andpreventing recurrent episodes. To achieve these goals, we need a safeand effective therapy that is well tolerated for long-term treatment.There are serious limitations to the long-term use of the currentlyapproved therapies. The most common, Lactulose, a non-absorbabledisaccharide, targets the gut flora responsible for ammonia production.It exerts its effects mainly by purging, with frequent bowel movements.Lactulose therapy relies on dose self-titration, aim is for 2-3 loosestools a day—unfortunately this goal is often exceeded. At tenunpredictable loose stools per day, leaving home—even for a short walkto the store—may become impossible or embarrassing. Patients go ondisability because of Lactulose rather than the HE it was prescribedfor. Severe diarrhea can cause dehydration and electrolyte abnormalitiesthat may even precipitate an HE episode. Nausea is not uncommon.Understandably, these factors can lead to poor adherence and limit longterm use.

Another approved treatment is neomycin. However, long-term use isseverely limited by its damaging side effects which includenephrotoxicity and sensorineural hearing loss—for which patients withadvanced liver disease are most susceptible. Not surprisingly, thesafety profile of neomycin is not conducive to long-term therapy. Giventhe limitations with both lactulose and neomycin—there clearly is anunmet medical need for a safe, effective, and well-tolerated long-termtherapy. Hepatic encephalopathy is a serious neurological complicationof advanced liver disease that disrupts quality of life, ability forself care and compliance, and results in frequent hospitalization

There are limited therapeutic options for HE and there remains an unmetmedical need for a safe, effective, and well-tolerated therapy forlong-term treatment. There has not been a new treatment for thisdebilitating disease for 30 years. Physicians have been sufficientlyimpressed with the efficacy, tolerability and safety data onrifaximin—and their favorable experience with this drug, even prior tothe exciting new trial data you will see today—to make rifaximin quitepossibly the most widely used antibiotic therapy for HE.

Study 3001 was designed to continuously monitor patients to ensure thevalidity and completeness of HE breakthrough capture. Followingscreening, subjects entered a treatment period that included weeklyvisits and/or phone calls with patients and caregivers. Subjects werefollowed for the protocol specified 168 days. Complete capture ofbreakthrough events as well as mortality and provided assurance of thevalidity of the study outcome. Narratives for each subject experiencingHE breakthrough, AE's resulting in termination, SAEs or death wereprovided in the NDA. Key entry criteria included:

-   -   Patients with advanced liver disease,    -   Presenting with at least 2 episodes of HE within 6 months of        screening; documented in medical records with a severity        equivalent to a Conn score≥2;    -   At both screening and baseline, subjects had a Conn score of 0        or 1, a MELD less than or equal to 25 and were required to have        a caregiver who assented to the patient's participation;    -   Patients were excluded if they had a condition that could        interfere with the protocol assessments, used alcohol within 14        days, sedatives within 7 days or evidence of current drug        dependence;

HESA combines both the clinical components of Conn andneuropsychological tests. Administration requires ˜45 minutes. It wasused as a tool to establish consistent scoring of Conn across studycenters. It provided a continuous reinforcement of standards anddefinitions

FIG. 17 succinctly covers the clinical assessments andneuropsychological testing of HESA. Rifaximin provided a significant,protective effect as demonstrated by a 58% reduction in the risk ofbreakthrough HE with a highly significant p-value. The benefit ofrifaximin is striking in that 78% of the patients now had zero eventsover 6 months. This is in contrast to the placebo group where only 54%maintain remission from HE. In a sick population who suffers fromfrequent adverse events, restricted living and a shortened life span,rifaximin is able to provide a meaningful benefit by preventingdeterioration in their mental status and motor skills.

There were a total of 104 events recorded from 299 participatingpatients. For the components, we are using descriptive statistics usingproportion analysis meeting the condition. 86 events, or 83% of thetotal events, consisted of patients experiencing a Conn Score of >=2,37% placebo and 20% rifaximin, resulting in a highly significantp-value.

Eighteen (18) events, or 17% of the total events, are included in thisnext category of patients experiencing a worsening of Conn and asterixisgrade of 1 each. 9% of placebo and 2% of rifaximin, also providing ahighly significant p-value.

Consistency of effect aids in determining whether the benefit is derivedfrom one or a few subgroups or if the effect is seen generally acrossall patient subgroups. Importantly, we tested for a treatment bysubgroup interaction to ascertain homogeneity in response acrosssubgroups. None of the subgroups tested for a significant interaction.Hazard ratios less than 1 indicate that the outcome favors rifaximin andgreater than 1 favors placebo. The result seen in all subgroupsconsistently reflect the clinical benefit in favor of rifaximin. Thisconsistency of outcome, coupled with the absence of a subgroup bytreatment interaction, support the robustness of the overall treatmenteffect.

This effect is maintained across subgroups of varying degrees ofseverity as it relates to MELD and Child-Pugh. Again, there is nosubgroup by treatment interaction here and the estimate of the treatmenteffect is approximately the same across all groups. In total, thesubgroup analyses demonstrate the remarkable consistency of the riskreduction seen in the Primary Endpoint analysis across all groups.

The analysis of the time to HE-related hospitalization results in a 50%reduction in risk with a significant p-value. A large proportion of HEepisodes resulted either in direct hospitalization or occurred duringthe hospitalization. It was shown that time to HE-caused hospitalization(defined as time to hospitalization directly resulting from HE), andtime to all-cause hospitalization were reduced with rifaximin, and theseanalyses show 56% and 30% reductions in risk respectively.

Other endpoints included, for example, the time to first worsening inConn or Asterixis Scores regardless of whether that change led to abreakthrough HE event; patient reported Quality of life, in particularfatigue, using the CLDQ; changes in blood ammonia, believed to be theprimary neurotoxin responsible for the HE; and the Critical flickerfrequency. The time to first time worsening in Conn Score reflects a 54%reduction in risk. Time to worsening of asterixis or hand flapping,shows a 35% reduction with a trending p-value. These data represent thechanges in each domain throughout the course of the trial. The resultsdemonstrate that rifaximin treated patients feel better. Thequestionnaire uses a 7-point Likert scale with 1=All the time and 7being none of the time. Thus, greater values represent better quality oflife. The change seen here in each subscale suggests a movement on eachscale of 1 category improvement over placebo. The changes we see inammonia and CFF are statistically significant and reflect improvement infavor of rifaximin. These results support the treatment effect ofrifaximin.

Analyses were undertaken to assess the sensitivity and specificity ofbreakthrough HE. Patients with lower CFF and fatigue assessments, and ahigher blood ammonia concentration had a greater likelihood ofexperiencing an HE breakthrough. These data provide further evidencethat the primary endpoint is objective and clinically meaningful. For3002, breakthrough HE data were collected to provide supportiveinformation regarding rifaximin's effect of preventing recurrence of HE.

Three populations were treated in study 3002, including,rifaximin-treated patients from Study 3001; crossover placebo-treatedpatients from Study 3001; and new HE patients.

Rifaximin subjects who maintained remission throughout 3001 demonstratedcontinued benefit during their participation in 3002. The incidence ofbreakthrough HE for rifaximin subjects was lower than the 3001 placebogroup demonstrating a 90% reduction in the risk of breakthrough HE. Notethat approximately 60% of these patients remain free of breakthroughafter almost 3 years. 82 placebo treated subjects from the 3001 studywere enrolled in 3002 and were followed for breakthrough. Once in theopen-label and receiving rifaximin, we see a 79% risk reduction comparedto their experience in the 3001 trial.

The all Rifaximin population demonstrates a 2.6-fold increase risk ofall-cause mortality for subjects who achieved a Conn score of at least2.

The following example will discuss new and novel aspects of rifaximin:

-   -   In vitro and in vivo pharmacological actions of rifaximin that        may contribute to its clinical benefit;    -   Rifaximin's ADME properties, including pharmacokinetics and its        excretory and metabolic fate; and    -   Drug-drug interaction studies.

Mechanistically, rifaximin binds to the beta-subunit of bacterial DNAdependent RNA polymerase resulting in inhibition of bacterial RNAsynthesis. In vivo, rifaximin ameliorates bacterial diarrheal symptomsand the majority of the dose is not absorbed and it concentrates in thegut, with high gut lumen concentrations, approximately 8000 μg/g ofstool. Interestingly, treatment of travelers' diarrhea occurs withoutsignificant alteration to the overall intestinal pathogen burden.

In vitro, rifaximin has multiple effects at subinhibitoryconcentrations, including, for example, increasing plasmid cured,reducing plasmid transfer, and reducing virulence.

We have observed effects of rifaximin on mammalian cells, including, forexample, detoxification pathways such as P-gp and 3A4 may be upregulatedin the gut. Rifaximin renders epithelial cells resistant to bacterialcolonization and internalization independent of the effects on bacteriaand reduces production and absorption of gut-derived neurotoxins, theprimary example being ammonia, which lead to HE in liver-impairedpatients.

In this example, 50 patients treated with rifaximin 1200 mg/day showedstatistically significant blood ammonia reduction. This reduction wasaccompanied by significant improvement in overall HE grade andindividual measures of HE. While discrete blood ammonia concentrationsmay be variable, serial measurements in individual patients have beenassociated with HE severity.

Rifaximin is a member of the rifamycin class of antibiotics. Thefunctional group shown in green differentiates rifaximin from otherrifamycins and leads to gut-specific activity.

Rifaximin is categorized as BCS 4; poorly soluble and poorly absorbed.It is also a substrate of P-glycoprotein, an efflux transporter. Theseproperties result in very low oral absorption. The small fraction thatis absorbed is cleared by three mechanisms: billiary, metabolic andrenal. Rifaximin undergoes first pass elimination via biliary excretionas unchanged rifaximin. There is one known metabolite; nearlyundetectable in healthy subjects, and very low in HE patients,approximately 2.5% of parent exposure. In both healthy and liver diseasesubjects, rifaximin renal clearance is <0.4%. Orally administeredrifaximin is eliminated almost entirely as unchanged rifaximin in thefeces. Steady-state rifaximin pharmacokinetics was examined in healthysubjects and in liver impaired subjects. Exposure is quite low in allpopulations studied.

In healthy volunteers, mean Cmax is less than 4 ng/mL. See FIG. 20 . Asliver impairment increases, AUC and Cmax increase correspondingly. Evenat their highest, exposures remain low, in the ng/mL range. Increasedexposure in liver impaired patients is well described in the literature,and may be attributed to several factors, including, for example,protein binding, reduced liver blood flow and reduced metaboliccapability. Limited access to the liver due to blood flow shuntingaround the liver, and reduced metabolism due to impaired hepatocyteenzyme activity, may reduce hepatic clearance. Either or both of thelatter two factors may be responsible for reducing clearance ofrifaximin and increasing exposure in liver impaired patients.

To put this exposure into further perspective see FIG. 21 which showsrifaximin data in comparison with other antibiotics, on a log scalebecause of the wide differences.

Patients with greatest liver impairment and highest plasma exposure haverifaximin levels more than 200-fold lower than those achieved with asystemic antibiotic, like rifampin—shown in blue. It's also more than10-fold lower than exposures observed with oral neomycin—shown here inpink—which is considered to be non-absorbed. Norfloxacin also is usedcommonly in this population, for SBP prophylaxis; it's a systemicantibiotic with plasma exposures greater than 35-fold higher thanrifaximin. The potential for rifaximin to cause drug-drug interactionswas explored here. Rifaximin does not significantly inhibit any majorP450 drug metabolizing enzyme, P-glycoprotein, or BSEP in subjects withnormal liver function. Knowing that other members of this class cancause interactions by upregulating important drug metabolizing enzymes,particularly CYP3A4, we examined the potential for this induction inclinical studies. Rifaximin's effect on midazolam, a classic CYP3A4substrate, was studied in healthy volunteers. After 16 days of rifaximin550 mg TID, a dose 50% higher than that used for HE, midazolam's AUC wasreduced by 10%. See FIG. 22 . In contrast, rifampin reduces midazolamAUC by 95% in similar experiments. This difference reflects not only anin vitro potency difference between rifampin and rifaximin, but an invivo disposition difference between the two compounds in terms ofrifaximin's low liver and systemic exposure. Based on these data we donot anticipate clinically significant drug interactions in subjects withnormal liver function. In summary, in vitro and in vivo data indicatethat rifaximin has bacteriostatic mechanisms as well as the ability toreduce bacterial adhesion and virulence. It lowers ammonia levels (SeeFIG. 23 ), which is linked to improvement in HE patients. The essentialdistinction between rifaximin and other rifamycins is its extremely lowsolubility and oral absorption, resulting in gut-targeted therapeuticeffects and limited systemic exposure. Although liver disease leads toincreased systemic exposure of rifaximin, the highest exposures seenwith rifaximin are substantially lower than what's observed with othersystemic and unabsorbed oral antibiotics. With this low systemicexposure comes a minimized drug-drug interaction risk.

Embodiments of the invention relate to all of the topical preparations,for instance ointments, pomades, creams, gels and lotions.

In solid dosage forms of rifaximin for oral administration (capsules,tablets, pills, dragees, powders, granules and the like), the activeingredient is typically mixed with one or morepharmaceutically-acceptable carriers, such as sodium citrate ordicalcium phosphate, and/or any of the following: (1) fillers orextenders, such as starches, lactose, sucrose, glucose, mannitol, and/orsilicic acid; (2) binders, such as, for example, carboxymethylcellulose,alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3)humectants, such as glycerol; (4) disintegrating agents, such asagar-agar, calcium carbonate, potato or tapioca starch, alginic acid,certain silicates, and sodium carbonate; (5) solution retarding agents,such as paraffin; (6) absorption accelerators, such as quaternaryammonium compounds; (7) wetting agents, such as, for example, acetylalcohol and glycerol monostearate; (8) absorbents, such as kaolin andbentonite clay; (9) lubricants, such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof; and (10) colouring agents. In the case ofcapsules, tablets and pills, the pharmaceutical compositions may alsocomprise buffering agents. Solid compositions of a similar type may alsobe employed as fillers in soft and hard-filled gelatin capsules usingsuch excipients as lactose or milk sugars, as well as high molecularweight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered activeingredient moistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions described herein, such as dragees, capsules, pills andgranules, may optionally be scored or prepared with coatings and shells,such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of rifaximin includepharmaceutically-acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active ingredient,the liquid dosage forms may contain inert diluents commonly used in theart, such as, for example, water or other solvents, solubilizing agentsand emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut,corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofurylalcohol, polyethylene glycols and fatty acid esters of sorbitan, andmixtures thereof.

In addition to inert diluents, the oral compositions can includeadjuvants such as wetting agents, emulsifying and suspending agents,sweetening, flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to rifaximin may contain suspending agents as,for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitoland sorbitan esters, microcrystalline cellulose, aluminum metahydroxide,bentonite, agar-agar and tragacanth, and mixtures thereof.

Pharmaceutical compositions for rectal or vaginal administration may bepresented as a suppository, which may be prepared by mixing rifaximinwith one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active agent.

Compositions which are suitable for vaginal administration also includepessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining such carriers as are known in the art to be appropriate.

Dosage forms for the topical or transdermal administration of rifaximinincludes powders, sprays, ointments, pastes, creams, lotions, gels,solutions, patches and inhalants. Rifaximin may be mixed under sterileconditions with a pharmaceutically-acceptable carrier, and with anypreservatives, buffers, or propellants which may be required.

Ointments, pastes, creams and gels may contain, in addition torifaximin, excipients, such as animal and vegetable fats, oils, waxes,paraffins, starch, tragacanth, cellulose derivatives, polyethyleneglycols, silicones, bentonites, silicic acid, talc and zinc oxide, ormixtures thereof.

Powders and sprays can contain, in addition to rifaximin, excipientssuch as lactose, talc, silicic acid, aluminium hydroxide, calciumsilicates and polyamide powder, or mixtures of these substances. Sprayscan additionally contain customary propellants, such aschlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, suchas butane and propane.

Rifaximin can be alternatively administered by aerosol. This isaccomplished by preparing an aqueous aerosol, liposomal preparation orsolid particles containing the compound. A non-aqueous (e.g.,fluorocarbon propellant) suspension could be used. Sonic nebulizers arepreferred because they minimize exposing the agent to shear, which canresult in degradation of the compound.

An aqueous aerosol is made, for example, by formulating an aqueoussolution or suspension of the agent together with conventionalpharmaceutically-acceptable carriers and stabilizers. The carriers andstabilizers vary with the requirements of the particular compound, buttypically include non-ionic surfactants (Tweens®, Pluronics®, orpolyethylene glycol), innocuous proteins like serum albumin, sorbitanesters, oleic acid, lecithin, amino acids such as glycine, buffers,salts, sugars or sugar alcohols. Aerosols generally are prepared fromisotonic solutions.

Pharmaceutical compositions suitable for parenteral administration maycomprise rifaximin in combination with one or morepharmaceutically-acceptable sterile isotonic aqueous or nonaqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain antioxidants, buffers,bacteriostats, solutes which render the formulation isotonic with theblood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and non-aqueous carriers which may beemployed in the pharmaceutical compositions include water, ethanol,polyols (such as glycerol, propylene glycol, polyethylene glycol, andthe like), and suitable mixtures thereof, vegetable oils, such as oliveoil, and injectable organic esters, such as ethyl oleate. Properfluidity can be maintained, for example, by the use of coatingmaterials, such as lecithin, by the maintenance of the required particlesize in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

In some cases, to prolong the effect of a drug, it is desirable to alterthe absorption of the drug. This may be accomplished by the use of aliquid suspension of crystalline, salt oramorphous material having poorwater solubility. The rate of absorption of the drug may then depend onits rate of dissolution which, in turn, may depend on crystal size andcrystalline form. Alternatively, delayed absorption of a drug form isaccomplished by dissolving or suspending the drug in an oil vehicle.

Injectable depot forms are made by forming microencapsule matrices ofrifaximin in biodegradable polymers such as polylactide-polyglycolide.Depending on the ratio of drug to polymer, and the nature of theparticular polymer employed, the rate of drug release can be controlled.Examples of other biodegradable polymers include poly(orthoesters) andpoly(anhydrides). Depot injectable formulations are also prepared byentrapping the drug in liposomes or microemulsions which are compatiblewith body tissue.

When the rifaximin is administered as a pharmaceutical, to humans andanimals, it can be given per se or as a pharmaceutical compositioncontaining, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) ofactive ingredient in combination with a pharmaceutically-acceptablecarrier.

Regardless of the route of administration selected rifaximin which maybe used in a pharmaceutical compositions of the present invention, isformulated into pharmaceutically-acceptable dosage forms by methodsknown to those of skill in the art.

Actual dosage levels and time course of administration of the activeingredients in the pharmaceutical compositions may be varied so as toobtain an amount of the active ingredient which is effective to achievethe desired therapeutic response for a particular subject, composition,and mode of administration, without being toxic to the subject. Anexemplary dose range is from 25 to 3000 mg per day.

XIFAXAN, a tradename for rifaximin, is approved for the following twouses:

1) Traveler's diarrhea: Rifaximin 200 mg are indicated for the treatmentof patients (≥12 years of age) with travelers' diarrhea caused bynoninvasive strains of Escherichia coli. Rifaximintablets should not beused in patients with diarrhea complicated by fever or blood in thestool or diarrhea due to pathogens other than Escherichia coli.

2) Hepatic encephalopathy: Rifaximin tablets 550 mg are indicated forthe maintenance of remission of hepatic encephalopathy in patients≥18years of age.

To reduce the development of drug-resistant bacteria and maintain theeffectiveness of rifaximin, and other antibacterial drugs, rifaximinwhen used to treat infection should be used only to treat or preventinfections that are proven or strongly suspected to be caused bysusceptible bacteria. When culture and susceptibility information areavailable, they should be considered in selecting or modifyingantibacterial therapy. In the absence of such data, local epidemiologyand susceptibility patterns may contribute to the empiric selection oftherapy.

XIFAXAN, a tradename for rifaximin, is approved for the following twouses:

1) Travelers' Diarrhea

Rifaximin 200 mg is indicated for the treatment of patients (≥12 yearsof age) with travelers' diarrhea caused by noninvasive strains ofEscherichia coli.

Rifaximin should not be used in patients with diarrhea complicated byfever or blood in the stool or diarrhea due to pathogens other thanEscherichia coli.

2) Hepatic Encephalopathy

Rifaximin 550 mg is indicated for reduction in risk of overt hepaticencephalopathy (HE) recurrence in patients≥18 years of age. In thetrials of rifaximin for HE, 91% of the patients were using lactuloseconcomitantly. Differences in the treatment effect of those patients notusing lactulose concomitantly could not be assessed.

Rifaximin has not been studied in patients with MELD (Model forEnd-Stage Liver Disease) scores>25, and only 8.6% of patients in thecontrolled trial had MELD scores over 19. There is increased systemicexposure in patients with more severe hepatic dysfunction.

Rifaximin can be administered orally with or without food. For treatmentof travelers' diarrhea patients should take one 200 mg tablet threetimes a day for 3 days. For hepatic encephalopathy patients should takeone 550 mg tablet two times a day.

Rifaximin tablets are contraindicated in patients with ahypersensitivity to rifaximin, any of the rifamycin antimicrobialagents, or any of the components in rifaximin tablets. Hypersensitivityreactions have included exfoliative dermatitis, angioneurotic edema, andanaphylaxis.

Rifaximin was not found to be effective in patients with diarrheacomplicated by fever and/or blood in the stool or diarrhea due topathogens other than Escherichia coli.

Discontinue rifaximin use if diarrhea symptoms get worse or persist morethan 24-48 hours and alternative antibiotic therapy should beconsidered.

Rifaximin is not effective in cases of travelers' diarrhea due toCampylobacter jejuni. The effectiveness of rifaximin in travelers'diarrhea caused by Shigella spp. and Salmonella spp. has not beenproven. Rifaximin should not be used in patients where Campylobacterjejuni, Shigella spp., or Salmonella spp. may be suspected as causativepathogens.

Clostridium difficile-associated diarrhea (CDAD) has been reported withuse of nearly all antibacterial agents, including rifaximin, and mayrange in severity from mild diarrhea to fatal colitis. Treatment withantibacterial agents alters the normal flora of the colon which may leadto overgrowth of C. difficile.

C. difficile produces toxins A and B which contribute to the developmentof CDAD. Hypertoxin producing strains of C. difficile cause increasedmorbidity and mortality, as these infections can be refractory toantimicrobial therapy and may require colectomy. CDAD must be consideredin all patients who present with diarrhea following antibiotic use.Careful medical history is necessary since CDAD has been reported tooccur over two months after the administration of antibacterial agents.

If CDAD is suspected or confirmed, ongoing antibiotic use not directedagainst C. difficile may need to be discontinued. Appropriate fluid andelectrolyte management, protein supplementation, antibiotic treatment ofC. difficile, and surgical evaluation should be instituted as clinicallyindicated.

Prescribing rifaximin for travelers' diarrhea in the absence of a provenor strongly suspected bacterial infection or a prophylactic indicationis unlikely to provide benefit to the patient and increases the risk ofthe development of drug-resistant bacteria.

There is increased systemic exposure in patients with severe hepaticimpairment. Animal toxicity studies did not achieve systemic exposuresthat were seen in patients with severe hepatic impairment. The clinicaltrials were limited to patients with MELD scores<25. Therefore, cautionshould be exercised when administering rifaximin to patients with severehepatic impairment (Child-Pugh C).

The safety of rifaximin 200 mg taken three times a day was evaluated inpatients with travelers' diarrhea consisting of 320 patients in twoplacebo-controlled clinical trials with 95% of patients receiving threeor four days of treatment with rifaximin. The population studied had amean age of 31.3 (18-79) years of which approximately 3% were ≥65 yearsold, 53% were male and 84% were White, 11% were Hispanic.

Discontinuations due to adverse reactions occurred in 0.4% of patients.The adverse reactions leading to discontinuation were taste loss,dysentery, weight decrease, anorexia, nausea and nasal passageirrigation.

All adverse reactions for rifaximin 200 mg three times daily thatoccurred at a frequency≥2% in the two placebo-controlled trials combinedare provided in Table 26. (These include adverse reactions that may beattributable to the underlying disease.)

The following adverse reactions, presented by body system, have alsobeen reported in <2% of patients taking rifaximin in the twoplacebo-controlled clinical trials where the 200 mg tablet was takenthree times a day for travelers' diarrhea. The following includesadverse reactions regardless of causal relationship to drug exposure:

TABLE 29 All Adverse Events With an Incidence ≥2% Among PatientsReceiving XIFAXAN Tablets, 600 mg/day, in Placebo-Controlled StudiesNumber (%) of Patients XIFAXAN Tablets, Placebo MedDRA Preferred Term600 mg/day (N = 320) N = 228 Flatulence  36 (11.3%)  45 (19.7%) Headache31 (9.7%) 21 (9.2%) Abdominal Pain NOS 23 (7.2%)  23 (10.1%) RectalTenesmus 23 (7.2%) 20 (8.8%) Defacation Urgency 19 (5.9%) 21 (9.2%)Nausea 17 (5.3%) 19 (8.3%) Constipation 12 (3.8%)  8 (3.5%) Pyrexia 10(3.1%) 10 (4.4%) Vomiting NOS  7 (2.2%)  4 (1.8%)

Blood and Lymphatic System Disorders: Lymphocytosis, monocytosis,neutropenia

Ear and Labyrinth Disorders: Ear pain, motion sickness, tinnitus

Gastrointestinal Disorders: Abdominal distension, diarrhea NOS, drythroat, fecal abnormality NOS, gingival disorder NOS, inguinal herniaNOS, dry lips, stomach discomfort

General Disorders and Administration Site Conditions: Chest pain,fatigue, malaise, pain NOS, weakness

Infections and Infestations: Dysentery NOS, respiratory tract infectionNOS, upper respiratory tract infection NOS

Injury and Poisoning: Sunburn

Investigations: Aspartate aminotransferase increased, blood in stool,blood in urine, weight decreased

Metabolic and Nutritional Disorders: Anorexia, dehydration

Musculoskeletal, Connective Tissue, and Bone Disorders: Arthralgia,muscle spasms, myalgia, neck pain

Nervous System Disorders: Abnormal dreams, dizziness, migraine NOS,syncope, loss of taste

Psychiatric Disorders: Insomnia

Renal and Urinary Disorders: Choluria, dysuria, hematuria, polyuria,proteinuria, urinary frequency

Respiratory, Thoracic, and Mediastinal Disorders: Dyspnea NOS, nasalpassage irritation, nasopharyngitis, pharyngitis, pharyngolaryngealpain, rhinitis NOS, rhinorrhea

Skin and Subcutaneous Tissue Disorders: Clamminess, rash NOS, sweatingincreased; and

Vascular Disorders: Hot flashes.

Hepatic Encephalopathy

The data described below reflect exposure to rifaximin 550 mg in 348patients, including 265 exposed for 6 months and 202 exposed for morethan a year (mean exposure was 364 days). The safety of rifaximin 550 mgtaken two times a day for reducing the risk of overt hepaticencephalopathy recurrence in adult patients was evaluated in a 6-monthplacebo-controlled clinical trial (n =140) and in a long term follow-upstudy (n=280). The population studied had a mean age of 56.26 (range:21-82) years; approximately 20% of the patients were ≥65 years old, 61%were male, 86% were White, and 4% were Black. Ninety-one percent ofpatients in the trial were taking lactulose concomitantly. All adversereactions that occurred at an incidence≥5% and at a higher incidence inrifaximin 550 mg-treated subjects than in the placebo group in thee6-month trial are provided in Table 27. (These include adverse eventsthat may be attributable to the underlying disease.)

TABLE 30 Adverse Events Occurring in ≥5% of Patients Receiving XIFAXANand at a Higher Incidence Than Placebo Table 40 Adverse Events Occuringin ≥5% of Patients Receiving XIFAXAN and at a Higher Incidence ThanPlacebo Number (%) of Patients XIFAXAN Tablets 550 mg BID Placebo MedDRAPreferred Term N = 140 N = 159 Edema peripheral 21 (15.0%) 13 (8.2%)Nausea 20 (14.3%)  12 (13.2%) Dizziness 18 (12.9%) 13 (8.2%) Fatigue 17(12.1%)  18 (11.3%) Ascites 16 (11.4%) 15 (9.4%) Muscle spasms 13(9.3%)  11 (6.9%) Pruritus 13 (9.3%)  10 (6.3%) Abdominal pain 12(8.6%)  13 (8.2%) Abdominal distension 11 (7.9%)  12 (7.5%) Anemia 11(7.9%)   6 (3.8%) Cough 10 (7.1%)  11 (6.9%) Depression 10 (7.1%)   8(5.0%) Insomnia 10 (7.1%)  11 (6.9%) Nasopharyngitis 10 (7.1%)  10(6.3%) Abdominal pain upper 9 (6.4%)  8 (5.0%) Arthralgia 9 (6.4%)  4(2.5%) Back pain 9 (6.4%) 10 (6.3%) Constipation 9 (6.4%) 10 (6.3%)Dyspnea 9 (6.4%)  7 (4.4%) Pyrexia 9 (6.4%)  5 (3.1%) Rash 7 (5.0%)  6(3.8%)

The following adverse reactions, presented by body system, have alsobeen reported in the placebo-controlled clinical trial in greater than2% but less than 5% of patients taking rifaximin 550 mg taken orally twotimes a day for hepatic encephalopathy. The following includes adverseevents occurring at a greater incidence than placebo, regardless ofcausal relationship to drug exposure.

Ear and Labyrinth Disorders: Vertigo;

Gastrointestinal Disorders: Abdominal pain lower, abdominal tenderness,dry mouth, esophageal variceal bleed, stomach discomfort.

General Disorders and Administration Site Conditions: Chest pain,generalized edema, influenza like illness, pain NOS

Infections and Infestations: Cellulitis, pneumonia, rhinitis, upperrespiratory tract infection NOS

Injury, Poisoning and Procedural Complications: Contusion, fall,procedural pain

Investigations: Weight increased

Metabolic and Nutritional Disorders: Anorexia, dehydration,hyperglycemia, hyperkalemia, hypoglycemia, hyponatremia

Musculoskeletal, Connective Tissue, and Bone Disorders: Myalgia, pain inextremity

Nervous System Disorders: Amnesia, disturbance in attention,hypoathesia, memory impairment, tremor

Psychiatric Disorders: Confusional state

Respiratory, Thoracic, and Mediastinal Disorders: Epistaxis; and

Vascular Disorders: Hypotension.

The following adverse reactions have been identified during postapproval use of rifaximin. Because these reactions are reportedvoluntarily from a population of unknown size, estimates of frequencycannot be made. These reactions have been chosen for inclusion due toeither their seriousness, frequency of reporting or causal connection torifaximin.

Infections and Infestations

Cases of C. difficile-associated colitis have been reported.

Hypersensitivity reactions, including exfoliative dermatitis, rash,angioneurotic edema (swelling of face and tongue and difficultyswallowing), urticaria, flushing, pruritus and anaphylaxis have beenreported. These events occurred as early as within 15 minutes of drugadministration.

In vitro studies have shown that rifaximin did not inhibit cytochromeP450 isoenzymes 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1 and CYP3A4 atconcentrations ranging from 2 to 200 ng/mL. Rifaximin is not expected toinhibit these enzymes in clinical use.

An in vitro study has suggested that rifaximin induces CYP3A4. However,in patients with normal liver function, rifaximin at the recommendeddosing regimen is not expected to induce CYP3A4. It is unknown whetherrifaximin can have a significant effect on the pharmacokinetics ofconcomitant CYP3A4 substrates in patients with reduced liver functionwho have elevated rifaximin concentrations.

An in vitro study suggested that rifaximin is a substrate ofP-glycoprotein. It is unknown whether concomitant drugs that inhibitP-glycoprotein can increase the systemic exposure of rifaximin.

Rifaximin was teratogenic in rats at doses of 150 to 300 mg/kg(approximately 2.5 to 5 times the clinical dose for travelers' diarrhea[600 mg/day], and approximately 1.3 to 2.6 times the clinical dose forhepatic encephalopathy [1100 mg/day], adjusted for body surface area).Rifaximin was teratogenic in rabbits at doses of 62.5 to 1000 mg/kg(approximately 2 to 33 times the clinical dose for travelers' diarrhea[600 mg/day], and approximately 1.1 to 18 times the clinical dose forhepatic encephalopathy [1100 mg/day], adjusted for body surface area).These effects include cleft palate, agnatha, jaw shortening, hemorrhage,eye partially open, small eyes, brachygnathia, incomplete ossification,and increased thoracolumbar vertebrae.

Reproduction studies have been performed in rats at doses up to 2.5 to5.0 times (adjusted for body surface area) the human dose, and inrabbits at doses up to 2.0 to 33.0 times (adjusted for body surfacearea) the human dose and have revealed no evidence of impaired fertilityor harm to the fetus due to rifaximin.

Two studies evaluated the pharmacokinetics of rifaximin in patients withhepatic impairment. In the first study mMean (SD) peak rifaximin plasmaconcentrations of 13.5 (14.8) ng/mL were detected in hepaticencephalopathy patients 3 hours after administration of the first doseof administered rifaximin 800 mg three times daily for 7 days; Llessthan 0.1% of the administered dose was recovered in urine after 7 days.Because of the limited systemic absorption of rifaximin, no specificdosing adjustments are recommended for patients with hepaticinsufficiency. In the second study, patients were administered rifaximin550 mg two times a day. Mean (SD) rifaximin steady-state systemicexposure values (Cmax) in those with hepatic impairment grades ofChild-Pugh A and Child-Pugh B were 19.5 (11.4) ng/mL and 25.1 (12.6)ng·h/mL (approximately 5.7- and 7.4-fold higher, respectively, thansteady-state Cmax values observed in healthy individuals). This increasein systemic exposure to rifaximin in patients with hepatic impairmentdoes not require a dosing adjustment with rifaximin due to itsgastrointestinal local action and low systemic bioavailability.

Exemplary dosages of contain rifaximin, a non-aminoglycosidesemi-synthetic, nonsystemic antibiotic derived from rifamycin SV.Rifaximin is a structural analog of rifampin. The chemical name forrifaximin is:(2S,16Z,18E,20S,21S,22R,23R,24R,25S,26S,27S,28E)-5,6,21,23,25-pentahydroxy-27-methoxy-2,4,11,16,20,22,24,26-octamethyl-2,7-(epoxypentadeca-[1,11,13]trienimino)benzofuro[4,5-e]pyrido[1,2-á]-benzimidazole-1,15(2H)-dione,25-acetate. The empirical formula is C₄₃H₅₁N₃O₁₁ and itsmolecular weight is 785.9.

Exemplary rifaximin tablets for oral administration are film-coated andcontain 200 mg or 550 mg of rifaximin. Each tablet contains colloidalsilicon dioxide, disodium edetate, glycerol palmitostearate,hypromellose, microcrystalline cellulose, propylene glycol, red ironoxide, sodium starch glycolate, talc, and titanium dioxide.

The dose-response relationship for rifaximin efficacy in reducing theseverity of hepatic encephalopathy (HE) was established in adouble-blind dose-ranging study (600, 1200, or 2400 mg total daily dosefor 7 days) in patients with Grade 1, 2, or 3 HE, improvements frombaseline were observed in all groups, as measured by an index measuringmultiple HE symptoms; mean changes (improvements) in symptom indexscores were −0.064, −0.103, and −0.107 in groups receiving total dailydoses of 600 mg, 1200 mg, and 2400 mg, respectively.

The mean plasma pharmacokinetic parameters of rifaximin in 14 healthysubjects after a single oral 400 mg dose given as 2×200 mg doses and asingle 550 mg dose in 12 healthy subjects under fed and fastingconditions are summarized in Table 31.

TABLE 31 Effect of Food on the Mean ± S.D. Pharmacokinetic ParametersSingle 400 mg Dose of Rifaximin (N = 14) Table 41 Effect of Food on theMean ± S.D. Pharmacokinetic Parameters Single 400 mg Dose of Single 550mg Dose of Rifaximin (N - 14) Rifaximin (N = 12) Parameter Fasting FedFasting Fed C_(max) (ng/mL) 3.80 ± 1.32 9.63 ± 5.93 4.04 ± 1.51 4.76 ±4.25 T_(max)(h) 1.21 ± 0.47 1.90 ± 1.52 0.75 (0.50-2.05)* 1.50(0.50-4.08)* Half-Life (h) 5.85 ± 4.34 5.95 ± 1.88 1.83 ± 1.38 4.84 ±1.34 AUC (ng · h/mL) 18.35 ± 9.48  34.70 ± 9.23  11.1 ± 4.15 22.5 ± 12.0

Rifaximin can be administered with or without food. Because systemicabsorption of rifaximin was low minimal in both the fasting state andwhen administered within 30 minutes of a high-fat breakfast, rifaximincan be administered with or without food.

14C-Rifaximin was administered as a single dose to 4 healthy malesubjects. The mean overall recovery of radioactivity in the urine andfeces of 3 subjects during the 168 hours after administration was96.94±5.64% of the dose. Radioactivity was excreted almost exclusivelyin the feces (96.62±5.67% of the dose), with only a small proportion ofthe dose (mean 0.32% of the dose) excreted in urine. Analysis of fecalextracts indicated that rifaximin was being excreted as unchanged drug.The amount of radioactivity in urine (<0.4% of the dose) suggests thatrifaximin is poorly absorbed from the gastrointestinal tract and isalmost exclusively and completely excreted in feces as unchanged drug.Mean rifaximin pharmacokinetic parameters were Cmax 4.3±2.8 ng/mL andAUCt 19.5±16.5 ng·h/mL with a median Tmax of 1.25 hours.

Travelers' Diarrhea

Systemic absorption of rifaximin (200 mg three times daily) wasevaluated in 13 subjects challenged with shigellosis on Days 1 and 3 ofa three-day course of treatment. Rifaximin plasma concentrations andexposures were low and variable. There was no evidence of accumulationof rifaximin following repeated administration for 3 days (9 doses).Peak plasma rifaximin concentrations after 3 and 9 consecutive dosesranged from 0.81 to 3.4 ng/mL on Day 1 and 0.68 to 2.26 ng/mL on Day 3.Similarly, AUC_(0-last) estimates were 6.95±5.15 ng·h/mL on Day 1 and7.83±4.94 ng·h/mL on Day 3. Rifaximin is not suitable for treatingsystemic bacterial infections because of limited systemic exposure afteroral administration.

Hepatic Encephalopathy

After a single dose and multiple doses of rifaximin 550 mg in healthysubjects, the mean time to reach peak plasma concentrations was about anhour. The pharmacokinetic (PK) parameters were highly variable and theaccumulation ratio based on AUC was 1.37.

The pharmacokinetics of patients with hepatic impairment (hepaticimpairment grades of Child-Pugh A and Child-Pugh B) taking rifaximin 550mg two times a day were evaluated in an open-label rifaximin study.Rifaximin exposure values (AUCτ) in subjects with Child-Pugh score A andB (118 and 161 ng*h/mL, respectively) were approximately 9.6- and13.1-fold higher than that observed in healthy subjects following twotimes a day oral doses of 550 mg (12.3 ng*h/mL), respectively.Intersubject variabilites in the pharmacokinetics of healthy subjectswere generally similar to those measured in subjects with hepaticimpairment.

Rifaximin can be administered with or without food.

Animal pharmacokinetic studies have demonstrated that 80% to 90% oforally administered rifaximin is concentrated in the gut with less than0.2% in the liver and kidney, and less than 0.01% in other tissues. Inadults with infectious diarrhea treated with rifaximin 800 mg daily forthree days, concentrations of rifaximin in stools averaged ˜8000 μg/gthe day after treatment ended.

In a mass balance study, after administration of 400 mg ¹⁴C-rifaximinorally to healthy volunteers, of the 96.94% total recovery, 96.62% ofthe administered radioactivity was recovered in feces almost exclusivelyas the unchanged drug and 0.32% was recovered in urine mostly asmetabolites with 0.03% as the unchanged drug. Rifaximin accounted for18% of radioactivity in plasma. This suggests that the absorbedrifaximin undergoes metabolism with minimal renal excretion of theunchanged drug. The enzymes responsible for metabolizing rifaximin areunknown.

In a separate study, rifaximin was detected in the bile aftercholecystectomy in patients with intact gastrointestinal mucosa,suggesting biliary excretion of rifaximin.

Hepatic Impairment

The systemic exposure of rifaximin was markedly elevated in patientswith hepatic impairment compared to healthy subjects. The mean AUC inpatients with Child-Pugh Class C hepatic impairment was 2-fold higherthan in patients with Child-Pugh Class A hepatic impairment (see Table28).

In vitro drug interaction studies have shown that rifaximin, atconcentrations ranging from 2 to 200 ng/mL, did not inhibit humanhepatic cytochrome P450 isoenzymes 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1,and 3A4. In an in vitro hepatocyte induction model, rifaximin was shownto induce cytochrome P450 3A4 (CYP3A4), an isoenzyme which rifampin isknown to induce. Two clinical drug-drug interaction studies usingmidazolam and an oral contraceptive containing ethinyl estradiol andnorgestimate demonstrated that rifaximin (200 mg TID for 3 days) did notalter the pharmacokinetics of these drugs, and rifaximin 550 mg TID for7 or 14 days resulted in only slightly reduced exposure to midazolamfollowing a single oral midazolam dose.

In an in vitro study, rifaximin was shown to induce CYP3A4 at theconcentration of 0.2 μM.

An in vitro study suggests that rifaximin is a substrate ofP-glycoprotein. In the presence of P-glycoprotein inhibitor verapamil,the efflux ratio of rifaximin was reduced greater than 50% in vitro. Theeffect of P-glycoprotein inhibition on rifaximin was not evaluated invivo.

The inhibitory effect of rifaximin on P-gp transporter was observed inan in vitro study. The effect of rifaximin on P-gp transporter was notevaluated in vivo.

The effect of rifaximin 200 mg administered orally every 8 hours for 3days and for 7 days on the pharmacokinetics of a single dose of eithermidazolam 2 mg intravenous or midazolam 6 mg orally was evaluated inhealthy subjects. No significant difference was observed in the metricsof systemic exposure or elimination of intravenous or oral midazolam orits major metabolite, 1′-hydroxymidazolam, between midazolam alone ortogether with rifaximin. Therefore, rifaximin was not shown tosignificantly affect intestinal or hepatic CYP3A4 activity for the 200mg three times a day dosing regimen.

After rifaximin 550 mg was administered three times a day for 7 days and14 days to healthy subjects, the mean AUC of single midazolam 2 mgorally was 3.8% and 8.8% lower, respectively, than when midazolam wasadministered alone. The mean C_(max) of midazolam was also decreased by4-5% when rifaximin was administered for 7-14 days prior to midazolamadministration. This degree of interaction is not considered clinicallymeaningful.

The effect of rifaximin on CYP3A4 in patients with impaired liverfunction who have elevated systemic exposure is not known.

Oral Contraceptives Containing 0.07 mg Ethinyl Estradiol and 0.5 mgNorgestimate

The oral contraceptive study utilized an open-label, crossover design in28 healthy female subjects to determine if rifaximin 200 mg orallyadministered three times a day for 3 days (the dosing regimen fortravelers' diarrhea) altered the pharmacokinetics of a single dose of anoral contraceptive containing 0.07 mg ethinyl estradiol and 0.5 mgnorgestimate. Results showed that the pharmacokinetics of single dosesof ethinyl estradiol and norgestimate were not altered by rifaximin.

In vitro study data suggest that rifaximin is a substrate forP-glycoprotein. Rifaximinis a weak inhibitor of P-gp; at concentrations(50 μM) significantly higher than those anticipated in plasma followingoral dose administration, rifaximin only partially inhibited transportof a model P-gp substrate. Therefore, no clinically significantinteractions with other drugs affected by P-glycoprotein areanticipated.

Rifaximin is excreted primarily in the feces. After oral administrationof 400 mg 14C398 rifaximin to healthy volunteers, approximately 97% ofthe dose was recovered in feces, almost entirely as unchanged drug, and0.32% was recovered in the urine.

Rifaximin is a non-aminoglycoside semi-synthetic antibiotic derived fromrifamycin SV; it is a structural analog of rifampin. The mechanism ofaction of rifaximin depends on the inhibition of DNA-dependent RNApolymerase of the target microorganisms, leading to the suppression ofinitiation of chain formation in RNA synthesis.

The lower rate of eradication of fecal pathogens in patients treatedwith rifaximin compared with fluoroquinolones and aminoglycosides andlack of alteration of gut flora indicate a unique mechanism of action.Rifaximin may alter virulence factors of enteric bacterial pathogenswithout killing them, as has been seen with subtherapeutic levels ofdrugs and colonization fimbriae of enterotoxigenic E. coli. Rifaximincaused morphological alterations in both susceptible and resistantbacterial strains at concentrations as low as 1/32 of the MIC.1Rifaximinreduced the viability and virulence of resistant bacteria, suggestingthat if in vivo pathogens are exposed to sub-MICs of the drug, not onlyare their physiological functions compromised, but gene virulence andantibiotic resistance are not fully expressed.

Rifaximin has in vitro antimicrobial activity against numerousGram-positive and Gram-negative bacteria, such as Escherichia coli.Animal and human studies demonstrate negligible systemic rifaximinabsorption (<1%) after oral administration. The negligible systemicabsorption of rifaximin from the gastrointestinal tract minimizes thepotential adverse events associated with systemically absorbedantibiotics. Rifaximin is delivered at high concentrations to thegastrointestinal tract, which is the therapeutic site of action.

Rifaximin acts by binding to the beta-subunit of bacterial DNA-dependentRNA polymerase resulting in inhibition of bacterial RNA synthesis.

Escherichia coli has been shown to develop resistance to rifaximin invitro. However, the clinical significance of such an effect has not beenstudied.

Rifaximin is a structural analog of rifampin. Organisms with highrifaximin minimum inhibitory concentration (MIC) values also haveelevated MIC values against rifampin. Cross-resistance between rifaximinand other classes of antimicrobials has not been studied.

Rifaximin has been shown to be active against the following pathogen inclinical studies of infectious diarrhea as described in herein.

For HE, rifaximin is thought to have an effect on the gastrointestinalflora.

In vitro susceptibility testing was performed according to the NationalCommittee for Clinical Laboratory Standards (NCCLS) agar dilution methodM7-A612. However, the correlation between susceptibility testing andclinical outcome has not been determined.

Escherichia coli has been shown to develop resistance to rifaximin invitro. However, the clinical significance of such an effect has not beenstudied. Rifaximin is a structural analog of rifampin. Organisms withhigh rifaximin minimum inhibitory concentration (MIC) values also haveelevated MIC values against rifampin. Cross resistance between rifaximinand other classes of antimicrobials has not been studied.

Malignant schwannomas in the heart were significantly increased in maleCrl:CD® (SD) rats that received rifaximin by oral gavage for two yearsat 150 to 250 mg/kg/day (doses equivalent to 2.4 to 4 times therecommended dose of 200 mg three times daily for travelers' diarrhea,and equivalent to 1.3 to 2.2 times the recommended dose of 550 mg twicedaily for hepatic encephalopathy, based on relative body surface areacomparisons). There was no increase in tumors in Tg.rasH2 mice dosedorally with rifaximin for 26 weeks at 150 to 2000 mg/kg/day (dosesequivalent to 1.2 to 16 times the recommended daily dose for travelers'diarrhea and equivalent to 0.7 to 9 times the recommended daily dose forhepatic encephalopathy, based on relative body surface areacomparisons).

The carcinogenic potential of rifaximin was examined in a 2 year studywith CD rats. Daily oral administration of at dose levels ranging from20, 50, to 250 mg/kg/day produced no evidence of a carcinogenic effect.

Similarly, in a study with Tg.rasH2 mice daily oral administration bygavage with rifaximin at doses up to 1500 mg/kg/day (males) and 2000mg/kg/day (females) for 26-weeks did not increase the incidence oftumors when compared to vehicle control.

Rifaximin was not genotoxic in the bacterial reverse mutation assay,chromosomal aberration assay, rat bone marrow micronucleus assay, rathepatocyte unscheduled DNA synthesis assay, or the CHO/HGPRT mutationassay. There was no effect on fertility in male or female rats followingthe administration of rifaximin at doses up to 300 mg/kg (approximately5 times the clinical dose of 600 mg/day, and approximately 2.6 times theclinical dose of 1100 mg/day, adjusted for body surface area).

Results from multiple-dose oral toxicity studies in rats, rabbits, anddogs showed negligible toxic effects of rifaximin at doses ranging from6 to 68 times the clinical dose for travelers' diarrhea (600 mg/day) fordurations of up to 39 weeks.

In a 26-week study with Tg.rasH2 mice orally administered rifaximin atdoses up to 1500 mg/kg/day (males) and 2000 mg/kg/day (females) 2/25female mice at 2000 mg/kg day presented ruffled fur and hunchedappearance in low incidence that did not reach statistical significance.

Oral administration of rifaximin for 3-6 months produced hepaticproliferation of connective tissue in rats (50 mg/kg/day) and fattydegeneration of liver in dogs (100 mg/kg/day). However, plasma druglevels were not measured in these studies. Subsequently, rifaximin wasstudied at doses as high as 300 mg/kg/day in rats for 6 months and 1000mg/kg/day in dogs for 9 months, and no signs of hepatotoxicity wereobserved. The maximum plasma AUC_(0-8 hr) values from the 6 month ratand 9 month dog toxicity studies (range: 42-127 ng·h/mL) was lower thanthe maximum plasma AUC_(0-8 hr) values in cirrhotic patients (range:19-306 ng·h/mL).

The efficacy of rifaximin given as 200 mg orally taken three times a dayfor 3 days was evaluated in 2 randomized, multi-center, double-blind,placebo-controlled studies in adult subjects with travelers' diarrhea.One study was conducted at clinical sites in Mexico, Guatemala, andKenya (Study 1). The other study was conducted in Mexico, Guatemala,Peru, and India (Study 2). Stool specimens were collected beforetreatment and 1 to 3 days following the end of treatment to identifyenteric pathogens. The predominant pathogen in both studies wasEscherichia coli.

The clinical efficacy of rifaximin was assessed by the time to return tonormal, formed stools and resolution of symptoms. The primary efficacyendpoint was time to last unformed stool (TLUS) which was defined as thetime to the last unformed stool passed, after which clinical cure wasdeclared. Table 32 displays the median TLUS and the number of patientswho achieved clinical cure for the intent to treat (ITT) population ofStudy 1. The duration of diarrhea was significantly shorter in patientstreated with rifaximin than in the placebo group. More patients treatedwith rifaximin were classified as clinical cures than were those in theplacebo group.

TABLE 32 Clinical Response in Study 1 (ITT population) XIFAXAN PlaceboEstimate (n = 125) (n = 129) (97.5% CI) P-Value Median 32.5 58.6 1.78^(a) 0.0002 TLUS (1.26, 2.50) (hours) Clinical 99 (79.2) 78 (60.5)18.7^(b) 0.001 cure, n (%) (5.3, 32.1) ^(a)Hazard Ratio ^(b)Differencein rates

Microbiological eradication (defined as the absence of a baselinepathogen in culture of stool after 72 hours of therapy) rates for Study1 are presented in Table 30 for patients with any pathogen at baselineand for the subset of patients with Escherichia coli at baseline.Escherichia coli was the only pathogen with sufficient numbers to allowcomparisons between treatment groups.

Even though rifaximin had microbiologic activity similar to placebo, itdemonstrated a clinically significant reduction in duration of diarrheaand a higher clinical cure rate than placebo. Therefore, patients shouldbe managed based on clinical response to therapy rather thanmicrobiologic response.

TABLE 33 Microbiologic Eradication Rates in Study 1 Subjects with aBaseline Pathogen Rifaximin Placebo Overall 48/70 (68.6) 41/61 (67.2) E.coli 38/53 (71.7) 40/54 (74.1)

The results of Study 2 supported the results presented for Study 1. Inaddition, this study also provided evidence that subjects treated withrifaximin with fever and/or blood in the stool at baseline had prolongedTLUS. These subjects had lower clinical cure rates than those withoutfever or blood in the stool at baseline. Many of the patients with feverand/or blood in the stool (dysentery-like diarrheal syndromes) hadinvasive pathogens, primarily Campylobacter jejuni, isolated in thebaseline stool.

Also in this study, the majority of the subjects treated with rifaximinwho had Campylobacter jejuni isolated as a sole pathogen at baselinefailed treatment and the resulting clinical cure rate for these patientswas 23.5% (4/17). In addition to not being different from placebo, themicrobiologic eradication rates for subjects with Campylobacter jejuniisolated at baseline were much lower than the eradication rates seen forEscherichia coli.

In an unrelated open-label, pharmacokinetic study of oral rifaximin 200mg taken every 8 hours for 3 days, 15 adult subjects were challengedwith Shigella flexneri 2a, of whom 13 developed diarrhea or dysenteryand were treated with rifaximin. Although this open-label challengetrial was not adequate to assess the effectiveness of rifaximin in thetreatment of shigellosis, the following observations were noted: eightsubjects received rescue treatment with ciprofloxacin either because oflack of response to rifaximin treatment within 24 hours (2), or becausethey developed severe dysentery (5), or because of recurrence ofShigella flexneri in the stool (1); five of the 13 subjects receivedciprofloxacin although they did not have evidence of severe disease orrelapse.

The efficacy of rifaximin 550 mg taken orally two times a day wasevaluated in a randomized, placebo-controlled, double-blind,multi-center 6-month trial of adult subjects from the U.S., Canada andRussia who were defined as being in remission (Conn score of 0 or 1)from hepatic encephalopathy (HE). Eligible subjects had ≥2 episodes ofHE associated with chronic liver disease in the previous 6 months.

A total of 299 subjects were randomized to receive either rifaximin(n=140) or placebo (n=159) in this study. Patients had a mean age of 56years (range, 21-82 years), 81% <65 years of age, 61% were male and 86%White. At baseline, 67% of patients had a Conn score of 0 and 68% had anasterixis grade of 0. Patients had MELD scores of either ≤10 (27%) or 11to 18 (64%) at baseline. No patients were enrolled with a MELD scoreof >25. Nine percent of the patients were Child-Pugh Class C. Lactulosewas concomitantly used by 91% of the patients in each treatment arm ofthe study. Per the study protocol, patients were withdrawn from thestudy after experiencing a breakthrough HE episode. Other reasons forearly study discontinuation included: adverse reactions (rifaximin 6%;placebo 4%), patient request to withdraw (rifaximin 4%; placebo 6%) andother (rifaximin 7%; placebo 5%).

The primary endpoint was the time to first breakthrough overt HEepisode. A breakthrough overt HE episode was defined as a markeddeterioration in neurological function and an increase of Conn score toGrade≥2. In patients with a baseline Conn score of 0, a breakthroughovert HE episode was defined as an increase in Conn score of 1 andasterixis grade of 1.

Breakthrough overt HE episodes were experienced by 31 of 140 subjects(22%) in the rifaximin group and by 73 of 159 subjects (46%) in theplacebo group during the 6-month treatment period. Comparison ofKaplan-Meier estimates of event-free curves showed rifaximinsignificantly reduced the risk of HE breakthrough by 58% during the6-month treatment period. Presented below in FIG. 24 is the Kaplan-Meierevent-free curve for all subjects (n=299) in the study.

When the results were evaluated by the following demographic andbaseline characteristics, the treatment effect of rifaximin 550 mg inreducing the risk of breakthrough overt HE recurrence was consistentfor: sex, baseline Conn score, duration of current remission anddiabetes. The differences in treatment effect could not be assessed inthe following subpopulations due to small sample size: non-White (n=42),baseline MELD>19 (n=26), Child-Pugh C (n=31), and those withoutconcomitant lactulose use (n=26).

FIG. 25 shows hazard ratios for the risk of experiencing breakthroughovert HE (rifaximin group divided by placebo group) for each subgroup,95% confidence intervals as determined by the Cox proportional hazardsmodel. P-values for differences between the rifaximin and placebo groupswere determined by log rank test.

HE-related hospitalizations were reported for 19 of 140 subjects (14%)and 36 of 159 subjects (23%) in the rifaximin and placebo groups,respectively. Rifaximin had a significant reduction of risk againstHE-related hospitalization during the 6-month treatment period; hazardratio in the rifaximin group relative to placebo was 0.500 (95% CI:0.287 to 0.873) (p=0.0129). Subjects in the rifaximin group had a 50%reduction in the risk of hospitalization due to HE during the 6-monthtreatment period when compared with placebo. See FIG. 26 : Time to FirstHE-Related Hospitalization in HE Study (up to 6 Months of Treatment, Day170) (ITT Population).

HE-related hospitalizations (hospitalizations directly resulting fromHE, or hospitalizations complicated by HE) were reported for 19 of 140subjects (14%) and 36 of 159 subjects (23%) in the rifaximin and placebogroups respectively. rifaximin had a significant reduction of riskagainst HE-related hospitalization during the 6-month treatment period;hazard ratio in the rifaximin group relative to placebo was 0.500 (95%CI: 0.287 to 0.873) (p=0.0129). Subjects in the rifaximin group had a50% reduction in the risk of hospitalization due to HE during the6-month treatment period when compared with placebo. See FIG. 26 : Timeto First HE-Related Hospitalization in HE Study (up to 6 Months ofTreatment, Day 170) (ITT Population).

Comparison of Kaplan-Meier estimates of event-free curves showedrifaximin significantly reduced the risk of HE-related hospitalizationsby 50% during the 6-month treatment period. Comparison of Kaplan-Meierestimates of event-free curves is shown in FIG. 25 .

Methods for dilution antimicrobial susceptibility tests for bacteriathat grow aerobically. National Committee for Clinical LaboratoryStandards, Sixth Edition, Wayne Pa. Approved Standard NCCLS DocumentM7-A6 January 2003; 23 (2).

Highly significant protective effects of rifaximin were observed withrespect to time to any increase from baseline in Conn score and time toany increase from baseline in asterixis grade when analyzedindependently; hazard ratio in the rifaximin group relative to placebowas 0.463 (95% CI: 0.312 to 0.685) (p<0.0001) for the risk ofexperiencing an increase in Conn score (ie, worsening in mental status)and 0.646 (95% CI: 0.414 to 1.008) (p=0.0523) for the risk ofexperiencing an increase in asterixis grade (ie, worsening in neuromotorfunctioning) during the 6-month treatment period.

Because lactulose was the most frequently used concomitant medicine, ananalysis was undertaken to analyze lactulose use at baseline and duringthe study to ensure that rifaximin treatment effect was not modified. Atbaseline and during the trial, lactulose use between the rifaximin andcontrol groups was no different. Thus, results of the study showingefficacy of rifaximin were not influenced by the use of lactulose.

In addition, patient subgroups were analyzed by MELD and Child-Pughanalyses to determine any differences between the treatment group andthe rifaximin group. It was found that the positive effects of rifaximinwere not limited by liver disease severity. In addition, it was alsofound that there was no significant interaction across subgroups.Accordingly, all these analyses demonstrated a risk reduction in favorof rifaximin. FIG. 28 illustrates that there was a consistency oftreatment affect across all the various subgroups that were administeredrifaximin.

Clostridium difficile-associated diarrhea (CDAD) has been reported withuse of nearly all antibacterial agents, including rifaximin, and mayrange in severity from mild diarrhea to fatal colitis. Treatment withantibiotics alters the normal flora of the colon which may lead to C.difficile. Patients can develop watery and bloody stools (with orwithout stomach cramps and fever) even as late as two or more monthsafter having taken the last dose of the antibiotic. If diarrhea occursafter therapy or does not improve or worsens during therapy, advisepatients to contact a physician as soon as possible.

Patients should be informed that in patients with severe hepaticimpairment (Child-Pugh C) there is an increase in systemic exposure torifaximin.

EXAMPLES

It should be appreciated that embodiments of the invention should not beconstrued to be limited to the examples, which are now described;rather, the invention should be construed to include any and allapplications provided herein and all equivalent variations within theskill of the ordinary artisan.

Example 1: Effects of Rifaximin on Subjects with Hepatic Insufficiency

Subjects were instructed to take one tablet of 550 mg of rifaximin bymouth 2 times per day—approximately every 12 hours. The rifaximin may beco-administered with other medications, for example, lactulose,antidepressants, anti-inflammatory, methadone, prescription andnon-prescription sleep aids (e.g., Lunesta™ (eszopiclone) and Ambien®(zolpidem tartrate)), and antihistamines, diuretics, laxatives or stoolsofteners, neurontin (gabapentin) and lyrica (pregabalin).

Lactulose use was optional for subjects. For subjects who usedlactulose, it was titrated to a dose during the 3 to 7-day observationperiod according to accepted medical practice.

Asterixis Grade

Asterixis (flapping tremor) was determined with the subject holding botharms and forearms extended with wrists dorsiflexed and fingers open for≥30 seconds. Asterixis was measured on a continuum of 5 grades, e.g.,grades 0 and 4=no abnormal movement vs. almost continuous flappingmotions, respectively as shown below:

Grade 0=No tremors;Grade 1=Rare flapping motions;Grade 2=Occasional, irregular flaps;Grade 3=Frequent flaps; andGrade 4=Almost continuous flapping motions.

Efficacy in regard to asterixis grade was measured as time to anyincrease from baseline in asterixis grade. Time to an increase inasterixis grade was computed as the number of days from the first doseof rifaximin to the initial occurrence of an increase from baseline inasterixis grade.

Breakthrough HE Episode

Relative risk of experiencing a breakthrough HE episode (e.g., Connscore Grade≥2, (e.g., 0 or 1 to ≥2) or a Conn and asterixis scoreincrease of 1 grade each) for each subject in the trial taking eitherrifaximin or the placebo was measured. The analysis compared time tofirst breakthrough HE episode for rifaximin versus placebo usingsurvival analysis methods. Time to first breakthrough HE episode wascomputed as the number of days from the first dose of rifaximin to theinitial occurrence of breakthrough HE (e.g., Conn score Grade≥2, or aConn and asterixis score increase of 1 grade each).

Change in mental status was measured by the Conn score (also known asthe West Haven score). The Conn score has been widely used as a measureof mental state in HE studies and is based on the criteria ofParsons-Smith as modified by Conn. The scale used in the Conn scoringsystem is described above.

Subjects had a Conn score of 0 or 1. An increase in the Conn score ofgreater than or equal to grade 2 was considered as a breakthrough HEepisode.

Hepatic Encephalopathy Scoring Algorithm (HESA)

The Hepatic Encephalopathy Scoring Algorithm (HESA) is a method thatuses both clinical and neuropsychological assessments to assess mentalstatus. The Algorithm has been validated previously and has beencorrelated with the Conn criteria.

The CFF test is recognized as a quantitative measure of CNS dysfunctionand that utilizes the correlation between cerebral processing ofoscillatory visual stimuli and its subsequent impairment due toincreased HE severity. The CFF test was administered, and statisticallysignificant greater improvement in CFF results were observed inrifaximin subjects when compared with placebo (p=0.0320).

The Critical Flicker Frequency (CFF) was assessed for each subject atscreening, baseline, visits 3 through 14 and the end of study visitusing the Lafayette Flicker Fusion (Lafayette Instrument Company, Inc).Circular light pulses with a 1:1 ratio between the visual impulse andthe interval were used with decreasing frequency in gradual steps of 0.5to 0.1 Hz/second. The frequency of the white light, which is initiallygenerated as a high-frequency pulse (50 Hz) and which gives the patientthe impression of a steady light, was reduced gradually until thepatient had the impression that the steady light had changed to aflicker. The patient registered this change by pressing a hand-heldswitch. The flicker frequencies were measured 8 times and from thesedata, the mean values for each patient were calculated. The process wasconducted in a quiet, semi-darkened room without distracting noises andtook about 10 minutes.

Critical Flicker Frequency Scores

The critical flicker frequency (CFF) was assessed for each subject usinga specialized CFF instrument. The CFF is the frequency at which thesubject observes a constant light transition to a flickering light andis measured in Hertz (Hz). CFF is an objective assessment of mentalstatus. A CFF value of 39 Hz has been shown to be the threshold forseparation between subjects who have manifest HE (e.g., Conn≥1) andthose without HE symptoms (e.g., Conn=0), with a lower CFF valueindicating more severe HE ⁽⁴³⁾.

The CFF was measured on a continuous scale and was the mean of 8separate fusion-to-flicker transition tests performed in rapidsuccession.

Ammonia Concentrations

Venous blood samples (10 mL) were collected and ammonia concentrationswere obtained by methods known in the art.

Time to increase from baseline in either the Conn score (mental stategrade) or asterixis grade

To analyze the time to a first breakthrough HE episode, survivalanalysis methods were used to assess the effectiveness of the rifaximintreatment on the time to increase from baseline in either the Conn score(mental state grade) or asterixis grade. Time to increase in either theConn score or asterixis grade was computed as the number of days fromthe first dose of rifaximin to the initial occurrence of either anincrease from baseline in Conn score or asterixis grade. The analysis oftime to increase in either Conn score or asterixis grade were based onthe comparison of time to event between rifaximin and placebo.

Time to First HE-related Hospitalization

The effect of rifaximin on time to first HE-related hospitalization wasdetermined. Time to first HE-related hospitalization was computed as thenumber of days from the first dose of rifaximin to the firsthospitalization for an HE related event. The analysis of time to firstHE-related hospitalization was based on the comparison of time tohospitalization between rifaximin and placebo.

Time to Development of Spontaneous Bacterial Peritonitis

The effect of rifaximin on time to development of spontaneous bacterialperitonitis (SBP) was determined. Time to development of SBP wascomputed as the number of days from the first dose of rifaximin to thetime of peritoneal fluid collection that resulted in a positive test forSBP. The analysis of time to development of SBP was based on thecomparison of time to event between rifaximin and placebo.

Mean Change From Baseline in Blood Ammonia Concentration and CriticalFlicker Frequency Values Over Time

Mean values and mean changes from baseline in blood ammoniaconcentration and critical flicker frequency values were collected.Analyses of blood ammonia concentrations and critical flicker frequencyvalues were based upon quantitative values (not qualitative grades).Treatment differences for mean change from baseline in these parameterswas estimated using a mixed effects model with fixed effects for timeand baseline value.

Mean Daily Lactulose Consumption Over Time

A subject's daily lactulose consumption was used to compute mean dailylactulose consumption for each month. Treatment differences for meanchange from baseline in mean daily lactulose consumption were estimated.

CLDQ

The CLDQ includes 29 items in the following six domains: abdominalsymptoms (three items), fatigue (five items), systemic symptoms (fiveitems), activity (three items), emotional function (eight items), andworry (five items). Summary scores for the CLDQ overall and each of thesix domains were computed and summarized at baseline and Days 28, 56,84, 112, 140 and 168 using descriptive statistics. Treatment differencesfor mean change in overall score and domain scores from baseline to Days28, 56, 84, 112, 140 and 168 were collected summarized and comparedbetween treatments.

Treatment differences for mean change from baseline to EOT weredetermined as the change from baseline at EOT in fatigue domain score ofChronic Liver Disease Questionnaire (CLDQ). Similarly, the mean changefrom baseline in blood ammonia concentration at EOT was also determined.

Assessment of Quality of Life

The SF-36, Chronic Liver Disease Questionnaire (CLDQ), and EpworthSleepiness Scale were used to measure health related quality of life.The 29 item CLDQ questionnaire consists of the following domains:fatigue, activity, emotional function, abdominal symptoms, systemicsymptoms, and worry.

Epworth Sleepiness Scale

Total scores for the Epworth Sleepiness Scale were computed andsummarized at baseline and Days 28, 56, 84, 112, 140 and 168 usingdescriptive statistics. Treatment differences for mean change in totalscores from baseline to Days 28, 56, 84, 112, 140 and 168 weresummarized and compared between treatments.

FIG. 1 is a line graph showing Lactulose daily use between subjectstaking placebos and subjects taking rifaximin as described above.

FIG. 2 is a line graph showing Kaplan Meier estimates of thedistribution of time to a breakthrough HE event for the placebo groupand the rifaximin group. As indicated there was an increased time tobreakthrough HE events for subjects taking rifaximin in comparison tosubjects taking the placebo.

FIG. 3 is a line graph showing Kaplan Meier estimates of thedistribution of time to a first HE related hospitalization. As indicatedthere was an increased time to hospitalization for subjects takingrifaximin in comparison to the placebo group.

FIG. 4 is a line graph showing Kaplan Meier estimates of thedistribution of time to a first increase in Conn scores. As indicatedthere was an increased time to the first increase in Conn scores forsubjects taking rifaximin in comparison to the placebo group.

FIG. 5 is a line graph showing Kaplan Meier estimates of thedistribution of time to a first increase in Asterixis grade. Asindicated there was an increased time to the first increase in Asterixisgrade for subjects taking rifaximin in comparison to the placebo group.

Example 2

The following tables provide further evidence supporting theadvantageous use of GI specific antibiotics, such as rifaximin, to treatsubjects suffering from HE.

TABLE 1 Time to Onset of Breakthrough HE Episode Placebo (N = 159) 550mg Rifaximin BID (N = 140) Cumulative Conditional Cumulative ConditionalAt Occurrences Occurrences Probability of At Occurrences OccurrencesProbability of Days Risk of Events of Events Events (SE) Survival Riskof Events of Events Events (SE) Survival [0-28) 158 20 20 0.13 (0.03)1.0000 140 13 13 0.09 (0.02) 1.0000 [28-56) 137 23 43 0.17 (0.03) 0.8734126 4 17 0.03 (0.02) 0.9071 [56-84) 113 14 57 0.12(0.03) 0.7262 120 6 230.05 (0.02) 0.8783 [84-140) 98 10 67 0.10(0.03) 0.6363 112 7 30 0.06(0.02) 0.8344 [140-168) 84 6 73 0.07 (0.03) 0.5713 98 1 31 0.01 (0.01)0.7820 >=168 38 0 73 0.00 (0.00) 0.5305 46 0 31 0.00 (0.00) 0.7740Harzard Ratio: 0.421 95% CI: (0.276, 0.641) p-value: <.0001

TABLE 2 Time to Onset of Breakthrough HE Episode by Baseline Conn ScoreLevel Placebo (N = 107) 550 mg Rifaximin BID (N = 93) CumulativeConditional Cumulative Conditional At Occurrences OccurrencesProbability of At Occurrences Occurrences Probability of Days Risk ofEvents of Events Events (SE) Survival Risk of Events of Events Events(SE) Survival [0-28) 107 13 13 0.12(0.03) 1.0000 93 11 11 0.12(0.03)1.0000 [28-56) 93 16 29 0.17 (0.04) 0.8779 81 3 14 0.04 (0.02) 0.8817[56-84) 77 7 36 0.09 (0.03) 0.7269 77 1 15 0.01 (0.01) 0.8491 [84-140)69 5 41 0.07 (0.03) 0.6608 75 3 18 0.04 (0.02) 0.8380 [140-168) 61 4 450.07 (0.03) 0.6129 68 1 19 0.01 (0.01) 0.8042 >=168 27 0 45 0.00 (0.00)0.5724 32 0 19 0.00 (0.00) 0.7924 Harzard Ratio: 0.441 95% CI: (0.258,0.754) p-value: 0.0028

TABLE 3 Time to Onset of Breakthrough HE Episode by Prior Lactulose UsePlacebo (N = 142) 550 mg Rifaximin BID (N = 123) Cumulative ConditionalCumulative Conditional At Occurrences Occurrences Probability of AtOccurrences Occurrences Probability of Days Risk of Events of EventsEvents (SE) Survival Risk of Events of Events Events (SE) Survival[0-28) 141 19 19 0.13 (0.03) 1.0000 123 12 12 0.10(0.03) 1.0000 [28-56)121 21 40 0.17(0.03) 0.8652 110 4 16 0.04 (0.02) 0.9024 [56-84) 100 1353 0.13 (0.03) 0.7151 104 5 21 0.05 (0.02) 0.8696 [84-140) 86 10 630.12(0.03) 0.6221 97 7 28 0.07 (0.03) 0.8278 [140-168) 73 5 68 0.07(0.03) 0.5498 84 1 29 0.01 (0.01) 0.7678 >=168 33 0 68 0.00 (0.00)0.5121 39 0 29 0.00 (0.00) 0.7586 Harzard Ratio: 0.424 95% CI: (0.274,0.655) p-value: 0.0001

TABLE 4 Time to Onset of First HE-Related Hospitalization Placebo (N =159) 550 mg Rifaximin BID Conditional (N = 140) Conditional CumulativeProbability Cumulative Probability At Occurrences Occurrences of AtOccurrences Occurrences of Events Days Risk of Events of Events Events(SE) Survival Risk of Events of Events (SE) Survival [0- 154 11 11 0.07(0.02) 1.0000 138 6 6 0.04 (0.02) 1.0000 28) [28- 131 14 25 0.11 (0.03)0.9286 125 4 10 0.03 (0.02) 0.9564 56) [56- 106 7 32 0.07 (0.02) 0.8293113 5 15 0.04 (0.02) 0.9258− 84) [84- 86 8 40 0.09 (0.03) 0.7743 100 520 0.05 (0.02) 0.8848 140) [140- 66 2 42 0.03 (0.02) 0.7023 86 3 23 0.04(0.02) 0.8402 168) >=168 30− 0 42 0.00 (0.00) 0.6810 39 0 23 0.00 (0.00)0.8108 Hazard Ratio: 0.521 958 CI: (0.313, 0.868) p-value: 0.0107

TABLE 5 Time to Any Increase from Baseline in Conn Score Placebo 550 mgRifaximin BID (N = 159) (N = 140) Cumulative Conditional CumulativeConditional At Occurrences Occurrences Probability of At OccurrencesOccurrences Probability of Days Risk of Events of Events Events (SE)Survival Risk of Events of Events Events (SE) Survival [0-28) 156 26 260.17 (0.03) 1.0000 139 17 17 0.12 (0.03) 1.0000 [28- 125 21 47 0.17(0.03) 0.8333 119 5 22 0.04 (0.02) 0.8777 56) [56- 100 15 62 0.15 (0.04)0.6928 109 9 31 0.08 (0.03) 0.8407 84) [84- 80 10 72 0.13 (0.04) 0.588394 5 36 0.05 (0.02) 0.7713 140) [140- 62 5 77 0.08 (0.03) 0.5143 79 0 360.00 (0.00) 0.7302 168) >=168 27 0 77 0.00 (0.00) 0.4729 37 1 37 0.03(0.03) 0.7302 Harzard Ratio: 0.463 95% CI: (0.312, 0.685) p-value:<.0001

TABLE 6 Time to Onset of Breakthrough HE Episode by Baseline MELD ScoreLevel Placebo 550 mg Rifaximin BID (N = 44) (N = 34) CumulativeConditional Cumulative Conditional At Occurrences OccurrencesProbability of At Occurrences Occurrences Probability of Days Risk ofEvents of Events Events (SE) Survival Risk of Events of Events Events(SE) Survival [0-28) 44 2 2 0.05 (0.03) 1.0000 34 1 1 0.03 (0.03) 1.0000[28- 42 4 6 0.10 (0.05) 0.9545 33 0 1 0.00 (0.00) 0.9706 56) [56- 38 1 70.03 (0.03) 0.8636 32 0 1 0.00 (0.00) 0.9706 84) [84- 37 3 10 0.08(0.04) 0.8409 32 1 2 0.03 (0.03) 0.9706 140) [140- 33 4 14 0.12 (0.06)0.7727 28 0 2 0.00 (0.00) 0.9398 168) >=168 14 0 14 0.00 (0.00) 0.679113 0 2 0.00 (0.00) 0.9398 Harzard Ratio: 0.171 95% CI: (0.039, 0.754)p-value: 0.0197

TABLE 7 Time to Onset of Breakthrough HE Episode by Baseline MELD ScoreLevel Placebo (N = 86) 550 mg Rifaximin BID Conditional (N = 85)Cumulative Probability Cumulative Conditional At Occurrences Occurrencesof Events At Occurrences Occurrences Probability of Days Risk of Eventsof Events (SE) Survival Risk of Events of Events Events (SE) Survival[0-28) 86 15 15 0.18 (0.04) 1.0000 85 8 8 0.09 (0.03) 1.0000 [28-56) 7013 28 0.19 (0.05) 0.8246 77 2 10 0.03 (0.02) 0.9059 [56-84) 56 11 390.20 (0.05) 0.6703 73 3 13 0.04 (0.02) 0.8822 [84- 45 7 46 0.16 (0.05)0.5387 68 6 19 0.09 (0.03) 0.8459 140) [140- 36 2 48 0.06 (0.04) 0.453958 1 20 0.02 (0.02) 0.7713 168) >=168 16 0 48 0.00 (0.00) 0.4284 27 0 200.00 (0.00) 0.7580 Harzard Ratio: 0.329 95% CI: (0.195, 0.556) p-value:<.0001

TABLE 8 Time to Onset of Breakthrough HE Episode by Baseline MELD ScoreLevel Placebo 550 mg Rifaximin BID (N = 14) (N = 11) CumulativeConditional Cumulative Conditional At Occurrences OccurrencesProbability of At Occurrences Occurrences Probability of Days Risk ofEvents of Events Events (SE) Survival Risk of Events of Events Events(SE) Survival [0-28) 14 3 3 0.21 (0.11) 1.0000 11 1 1 0.09 (0.09) 1.0000[28- 11 4 7 0.36 (0.15) 0.7857 10 0 1 0.00 (0.00) 0.9091 56) [56- 7 2 90.29 (0.17) 0.5000 10 3 4 0.30 (0.14) 0.9091 84) [84- 5 0 9 0.00 (0.00)0.3571 7 0 4 0.00 (0.00) 0.6364 140) [140- 4 0 9 0.00 (0.00) 0.3571 7 04 0.00 (0.00) 0.6364 168) >=168 2 0 9 0.00 (0.00) 0.3571 3 0 4 0.00(0.00) 0.6364 Harzard Ratio: 0.403 95% CI: (0.123, 1.313) p-value:0.1315

TABLE 9 Time to Onset of Breakthrough HE Episode by Prior Lactulose UsePlacebo 550 mg Rifaximin BID (N = 134) (N = 127) Cumulative ConditionalCumulative Conditional At Occurrences Occurrences Probability of AtOccurrences Occurrences Probability of Days Risk of Events of EventsEvents (SE) Survival Risk of Events of Events Events (SE) Survival [0-134 18 18 0.13 (0.03) 1.0000 127 12 12 0.09 (0.03) 1.0000 28) [28- 11520 38 0.17 (0.04) 0.8652 114 4 16 0.04 (0.02) 0.9055 56) [56- 95 14 520.15 (0.04) 0.7147 108 6 22 0.06 (0.02) 0.8737− 84) [84- 80 9 61 0.11(0.04) 0.6094 100 6 28 0.06 (0.02) 0.8252 140 [140- 68 5 66 0.07 (0.03)0.5408 88 1 29 0.01 (0.01) 0.7754 168) >=168 31 0 66 0.00 (0.00) 0.501141 0 29 0.00 (0.00) 0.7666 Hazard Ratio: 0.399 95% CI: (0.258, 0.618)p-value: <.0001

TABLE 10 Time to Any Increase from Baseline in Asterixis Grade Placebo550 mg Rifaximin BID (N = 159) (N = 140) Cumulative ConditionalCumulative Conditional At Occurrences Occurrences Probability of AtOccurrences Occurrences Probability of Days Risk of Events of EventsEvents (SE) Survival Risk of Events of Events Events (SE) Survival[0-28) 154 20 20 0.13 (0.03) 1.0000 137 13 13 0.10 (0.03) 1.0000 [28-120 15 35 0.13 (0.03) 0.8697 116 7 20 0.06 (0.02) 0.9048 56) [56- 91 439 0.04 (0.02) 0.7610 101 7 27 0.07 (0.03) 0.8499 84) [84- 76 6 45 0.08(0.03) 0.7275 87 3 30 0.03 (0.02) 0.7910 140) [140- 61 4 49 0.07 (0.03)0.6701 74 1 31 0.01 (0.01) 0.7637 168) >=168 27 1 50 0.04 (0.04) 0.626234 1 32 0.03 (0.03) 0.7534 Harzard Ratio: 0.646 95% CI: (0.414, 1.008)p-value: 0.0523

TABLE 11 Mean Change from Baseline in Blood Ammonia Concentration(μg/dL) 550 mg Placebo Rifaximin BID Assessment Time (N = 159) (N = 140)P-value Day 28 n 126 121 Mean 89.3 88.4 SD 48.19 49.02 Median 87.0 74.0Min 2 25 Max 315 326 Change from Baseline to Day 28 n 117 117 0.6268Mean −1.1 −2.1 SD 48.32 44.37 Median 1.0 −2.0 Min −252 −164 Max 133 176

TABLE 12 Mean Change from Baseline in Critical Flicker Frequency Test(Hz) Placebo 550 mg Rifaximin Assessment Time (N = 159) BID (N = 140)P-value Day 140 n 70 87 Mean 38.7 38.7 SD 5.47 4.76 Median 38.8 38.9 Min26 27 Max 50 49 Change from Base- line to Day 140 n 70 87 0.0266 Mean1.1 1.4 SD 4.10 4.84 Median 0.9 1.5 Min −12 −15 Max 12 12

TABLE 13 Mean Change from Baseline in Critical Flicker Frequency Test(Hz) Placebo 550 mg Rifaximin Assessment Time (N = 159) BID (N = 140)P-value EOT n 155 139 Mean 37.6 37.8 SD 5.98 4.88 Median 37.9 37.8 Min21 25 Max 50 49 Change from Base- line to EOT n 155 139 0.0320 Mean 0.40.9 SD 4.70 4.75 Median 0.2 0.1 Min −12 −14 Max 16 11

TABLE 14 Number of Subjects in Each Level of Change from Baseline inConn Score by Treatment Group 550 mg Odds Ratio Assessment PlaceboRifaximin BID (550 mg Rifaximin 95% CI for Time Statistics (N = 159) (N= 140) BID/Placebo) Odds Ratio P-value Change from Baseline to EOT −1 n(%) 18 (11.5%) 26 (18.7%) 2.46 (1.49, 4.09) 0.0005 0 n (%) 100 (63.7%) 101 (72.7%)  1 n (%) 29 (18.5%) 10 (7.2%)  2 n (%) 9 (5.7%) 2 (1.4%) 3 n(%) 1 (0.6%) 0 n 157 139 Mean 0.2 −0.1 SD 0.74 0.56 Median 0.0 0.0 Min−1 −1 Max 3 2

TABLE 15 Number of Subjects in Each Level of Change from Baseline inAsterixis Grade by Treatment Group Odds Ratio 550 mg (550 mg AssessmentPlacebo Rifaximin BID Rifaximin 95% CI for Time Statistics (N = 159) (N= 140) BID/Placebo) Odds Ratio P-value Change from Baseline to EOT −2 n(%) 1 (0.6%)  1 (0.7%) 1.88 (1.10, 3.23) 0.0207 −1 n (%) 14 (8.9%)   18(12.9%) 0 n (%) 114 (72.6%)  108 (77.7%) 1 n (%) 18 (11.5%) 10 (7.2%) 2n (%) 8 (5.1%)  2 (1.4%) 3 n (%) 1 (0.6%) 0 4 n (%) 1 (0.6%) 0 n 157 139Mean 0.2 0.0 SD 0.76 0.54 Median 0.0 0.0 Min −2 −2 Max 4 2

TABLE 16 Mean Change from Baseline for Epworth Sleepiness Total ScorePlacebo 550 mg Rifaximin Assessment Time (N = 159) BID (N = 140) P-valueDay 28 N 91 87 Mean 9.1 10.0 SD 4.84 5.51 Median 8.0 9.0 Min 0 0 Max 2123 Change from Base- line to Day 28 N 90 86 0.0593 Mean −1.1 −0.2 SD4.79 3.53 Median −1.0 0.0 Min −17 −14 Max 14 7

TABLE 17 Time to Onset of First HE-Related Hospitalization Placebo 550mg Rifaximin BID (N = 159) (N = 140) Cumulative Conditional CumulativeConditional At Occurrences Occurrences Probability of At OccurrencesOccurrences Probability of Days Risk of Events of Events Events (SE)Survival Risk of Events of Events Events (SE) Survival [0-28) 155 11 110.07 (0.02) 1.0000 139 4 4 0.03 (0.01) 1.0000 [28- 132 12 23 0.09 (0.03)0.9288 130 4 8 0.03 (0.02) 0.9711 56) [56- 108 7 30 0.06 (0.02) 0.8440119 4 12 0.03 (0.02) 0.9411 84) [84- 88 4 34 0.05 (0.02) 0.7893 106 5 170.05 (0.02) 0.9094 140) [140- 72 2 36 0.03 (0.02) 0.7535 92 2 19 0.02(0.02) 0.8665 168) >=168 34 0 36 0.00 (0.00) 0.7325 43 0 19 0.00 (0.00)0.8475 Harzard Ratio: 0.500 95% CI: (0.287, 0.873) p-value: 0.0129

Example 3: CYP3A4 is Not Induced by Rifaximin

Induction of CYP3A4 by rifaximin was observed based on decreasedmidazolam AUC by ˜25%. A higher systemic exposure is expected in amajority of the target patient population.

When rifaximin was orally administered at high doses (1650 mg/day) forat least 7 days, the mean C_(max), AUC_(0-t), and AUC_(0-∞)of midazolamwere reduced by <25%. Rifaximin is a potential CYP3A4 inducer, in vitrostudies have shown it to have a lower induction potency than rifampin.(The estimated intestinal lumen concentration of rifaximin isapproximately 5 μM. In the in vitro study, CYP3A4 activity was induced1.7-fold and 1.8-fold at rifaximin 1 μM and 10 μM; at the sameconcentrations, rifampin induced CYP3A4 3.7-fold and 4-fold,respectively. Furthermore, rifaximin's gut-targeted distribution isbelieved to limit its CYP3A4 induction mechanism to the intestine,sparing hepatic induction as a result of low systemic exposure. That is,there is a separation of intestinal and hepatic induction for rifaximinThis is shown in studies disclosed herein in humans receiving rifaximin,as supported by the absence of induction when either intravenous or oralmidazolam was administered following rifaximin 200 mg TID for up to 7.

Without wishing to be bound by any particular scientific theory, it isthought that any risk of hepatic CYP3A4 induction likely is furthermitigated in hepatically impaired patients, for whom significantfractions of portal blood flow are shunted around the liver;³ therefore,their increased systemic exposure should be accompanied by aproportional decrease in exposure to hepatocytes and the patients shouldincur no net increase in risk of hepatic CYP3A4 induction.

Example 4: Drug Interaction Studies

Two clinical drug-drug interaction studies were conducted with therifaximin 200 mg tablet and one drug-drug interaction study with the 550mg tablet. Two studies using midazolam, a known substrate for CYP3A4,and 1 study using an oral contraceptive containing ethinyl estradiol andnorgestimate were conducted to assess the effect of rifaximin on thepharmacokinetics of these drugs. Based on the results of these studiesand in vitro induction and inhibition studies using human liverfractions, no clinically relevant drug interactions are anticipated withrifaximin.

Although in vitro studies demonstrated the potential of rifaximin tointeract with cytochrome P450 3A4 (CYP3A4), a clinical drug-druginteraction study demonstrated that rifaximin did not significantlyaffect the pharmacokinetics of midazolam either presystemically orsystemically. An additional clinical drug-drug interaction study showedno effect of rifaximin on the presystemic metabolism of an oralcontraceptive containing ethinyl estradiol and norgestimate. Therefore,clinical interactions with drugs metabolized by human cytochrome P450isoenzymes are not expected.

Two studies have been performed to evaluate the potential for druginteractions with midazolam. The first was an open-label, randomized,crossover, drug-interaction trial designed to assess the effect ofrifaximin 200 mg administered orally (PO) every 8 hours (Q8H) for 3 daysand every 8 hours for 7 days, on the pharmacokinetics of a single doseof either midazolam 2 mg intravenous (IV) or midazolam 6 mg PO. Nosignificant difference was observed in the metrics of systemic exposureor elimination of IV or PO midazolam or its major metabolite,1′-hydroxymidazolam, between midazolam alone or together with rifaximin.Therefore, rifaximin was not shown to significantly affect intestinal orhepatic CYP3A4 activity.

The second study, an open-label, drug-interaction study examined theeffect of rifaximin, 550 mg three times daily, on orally administered(PO) midazolam 2 mg when dosed for 7 and 14 consecutive days. In thisstudy rifaximin was shown to be a weak inducer of CYP3A4; given the lowsystemic exposure of rifaximin, this interaction is believed to belimited to the gastrointestinal tract. This induction is both dose- anddosing-duration dependent. When rifaximin was orally administered athigh doses (1650 mg/day) for at least 7 days, the mean C_(max),AUC_(0-t), and AUC_(0-∞) of midazolam were reduced by <25%.

In vitro hERG potency and in vitro protein binding of rifaximin. In thein vitro hERG studies, rifaximin concentrations up to 300 μM failed toachieve 50% inhibition of the hERG potassium current. Due to rifaximinprecipitation at 300 μM, the IC₅₀ was estimated to be greater than 100μM. In fact, 50% inhibition could not be achieved; at 100 μM, meaninhibition was 34.5%. The highest C_(max) observed in a hepaticallyimpaired patient in a study was 52.2 ng/mL (0.0664 μM); the highest freefraction observed in a subset of plasma samples from patients enrolledin this study was 44.7%. Using these numbers, the highest anticipatedfree plasma exposure would be 0.03 μM, which represents a reduction of≥3000-fold in comparison with the highest concentration at whichrifaximin could be tested in the hERG experiments. This safety margingreatly exceeds the 30-fold separation between hERG IC₅₀ and unboundC_(max) that is commonly associated with minimization of risk ofclinical QT prolongation.

Example 5: Time to First Breakthrough Event

An efficacy parameter for a first study was the occurrence of an episodeof breakthrough overt HE during treatment. Breakthrough overt HEepisodes were measured by using the Conn score (or West Haven grade),and the asterixis grade. A breakthrough overt HE episode, as defined forthe first study, was a marked, clinically significant deterioration inneurological function that can result in a deleterious effect on selfcare, and lead to hospitalization. The efficacy endpoint, time to firstbreakthrough overt HE episode, showed a highly significant protectiveeffect of rifaximin (p<0.0001 for between-group difference in relativerisk). Rifaximin treatment resulted in a 57.9% reduction, when comparedwith placebo, in the risk of experiencing breakthrough overt HE duringthe 6-month treatment period.

In addition, this study also showed that the time to first breakthroughovert HE also showed a highly significant protective effect of rifaximinwhen analyzed in separate geographic regions, North America versusRussia.

Rifaximin treatment results in fewer overt HE episodes that mayotherwise incapacitate the patient, may alleviate the burden on familymembers who are required to care for the patient, and reduces the burdenof hospitalization in this patient population and the healthcare system.

In a second study, similar results were shown, for example, the secondstudy with respect to time to first breakthrough overt HE episode: theKaplan-Meier estimates of time to first breakthrough overt HE episodewere similar between the rifaximin group in the first study and newrifaximin subjects in this second study . Also, similar proportions ofsubjects had breakthrough overt HE in the rifaximin group of the firststudy (22%, 31 of 140 [rifaximin group]) and in the new rifaximin groupof the second study (27.6%, 54 of 196).

Additionally, when the first study placebo subjects crossed over torifaximin therapy by entering the second study, a protective effect ofrifaximin was observed: the first study a 70% reduction in risk ofexperiencing breakthrough overt HE during rifaximin treatment in thesecond study when compared with their prior placebo experience in thefirst study. This reduction took place in spite of the aging andpresumably progressing nature of the population with chronic liverdisease.

The second study also showed that the protective effect of rifaximin wasdurable: the estimate of time-to-first breakthrough HE demonstratedlong-term maintenance of remission from breakthrough HE when rifaximinsubjects in remission after participation in the first study werefollowed in the second study (up to 680 days of rifaximin therapy;median exposure durations were 168 days in the first study and 253 daysin the second study). The incidence of breakthrough HE episode for theserifaximin subjects relative to the first study placebo was lower, anindication of fewer breakthrough HE episodes with rifaximin treatment.

A critical flicker frequency (CFF) assessment, a recognized quantitativemeasure of CNS dysfunction, was an efficacy endpoint in the first study.CFF tests utilize the correlation between cerebral processing ofoscillatory visual stimuli and CNS impairment due to increased HEseverity. This test identifies a frequency at which a flickering lightis perceived as steady. A decline in this frequency has been associatedwith increasing severity of HE. Likewise, elevation in blood ammonia,another endpoint in the first study, is a quantitative assessmentassociated with the CNS effects underlying overt HE.

Comparisons of changes from baseline to end of study in CFF results andin venous ammonia levels showed statistically significant, greaterimprovement over the course of the study in the rifaximin group whencompared to placebo (p=0.0320 for CFF changes and p=0.0391 for venousammonia changes). In the first study, a correlation between CFF resultsand breakthrough overt HE (primary efficacy measure) was noted. Venousammonia levels were found to be correlated to the occurrence ofbreakthrough overt HE in the first study.

Results for other efficacy endpoints also demonstrated protectiveeffects of rifaximin. In particular, the other efficacy endpoint of timeto first HE-related hospitalization showed a reduction in risk forrifaximin subjects.

In the first study, the analysis of time to first HE-relatedhospitalization (e.g., hospitalization directly resulting from HE orhospitalization complicated by HE) demonstrated that the reduction inrisk of hospitalization due to HE was 50% in the rifaximin group, whencompared with placebo, during the 6-month treatment period. TheHE-related hospitalization rate was 0.38 event/person exposure years(PEY), rifaximin versus 0.78 event/PEY, placebo after normalization toexposure.

In the first study, the risk of HE-caused hospitalization (e.g.,hospitalization directly resulting from HE only) was reduced by 56% inthe rifaximin group when compared with placebo. The HE-causedhospitalization rate was 0.30 events/PEY in the rifaximin group versus0.72 event/PEY in the placebo group.

In the first study, the risk of all-cause hospitalization rate wasreduced by 30% in the rifaximin group when compared to placebo. Theall-cause hospitalization rate was 0.92 events/PEY in the rifaximingroup versus 1.31 event/PEY in the placebo group.

In the second study, the low HE-caused hospitalization rate wasmaintained at rates consistent with those in the first study: HE-causedhospitalization rate was 0.29 event/PEY and all cause hospitalization inthe second study was 0.66 event/PEY. The consistently lowHE-related/HE-caused hospitalization rate in rifaximin-treated subjectsin the first study and in the second study was at least partly a resultof maintaining remission from demonstrated HE in subjects with end-stageliver disease.

Hepatic encephalopathy is associated with a low quality of life comparedto age-matched patients without HE. Patients with HE experience symptomsincluding fatigue, daytime sleepiness, and lack of awareness (Conn score1); and confusion and disorientation (Conn score 2) that significantlyinterfere with day-to-day function and decreased ability for self care.Often, this lack of self care can lead to improper nutrition andnon-adherence to therapy and can further escalate into more severesymptoms such as increased somnolence, gross disorientation and stupor,which require hospitalization. Rifaximin treatment protects against HErelated/caused hospitalization, thereby improving the functional statusfor the patient and benefitting his/her caregiver; and reducing theeconomic cost related to liver cirrhosis and associated HE.

There are limited treatment options in the United States for patientswith recurrent HE. Neomycin sulfate is only approved for the adjunctivetherapy in hepatic coma. Conventional therapy aims to lower theproduction and absorption of ammonia. Nonabsorbable disaccharides, eg,lactulose or lactitol, are typically used as first-line therapy for HE.There is evidence that nonabsorbable disaccharides lower plasma levelsof ammonia by changing nitrogen metabolism in colonic flora andincreasing fecal excretion of nitrogen. Broadspectrum, GI-activeantibiotics including neomycin, metronidazole, vancomycin, andparomomycin have been used with or without lactulose. These antibioticsappear to act indirectly by inhibiting the splitting of urea bydeaminating bacteria, thus reducing the production of ammonia and otherpotential toxins. Current guidelines recommend (not FDA approved)antibiotic therapy with neomycin or metronidazole as an alternative totreatment with nonabsorbable disaccharides.

Common side effects of nonabsorbable disaccharide (e.g., lactulose)therapy include an unpleasant taste that can hinder treatmentcompliance, a dosing schedule that is linked to bowel habits, and GIside effects such as bloating, abdominal cramps, and diarrhea. Diarrhearesulting in dehydration has been reported with the use of lactulose, asignificant consequence for patients with HE as electrolyteabnormalities can worsen HE and lead to renal dysfunction.

The use of systemically absorbed antibiotics such as neomycin in thetreatment of HE is hampered by ototoxicity and nephrotoxicity associatedwith long-term use. The incidence of aminoglycoside-inducednephrotoxicity is substantially greater in patients with advanced liverdisease than in patients without liver disease. The frequency ofaminoglycoside-induced nephrotoxicity in the general population is 3% to11%. Leitman reported that nephrotoxicity occurred in 73% of patientswith liver disease versus 34% of patients without liver disease whoreceived aminoglycosides by intravenous administration duringhospitalization; and Cabrera reported that renal tubular damage orfunctional renal impairment was observed in 60% ofaminoglycoside-treated cirrhotic patients (intravenous administrationduring hospitalization). Additionally, a high mortality rate andsustained renal damage were noted in cirrhotic patients who developedaminoglycoside-induced renal tubular damage. Therefore, aminoglycosidesare now widely considered as contraindicated in patients with advancedliver disease.

Rifaximin is an attractive therapy for the treatment of patients with HEbecause of its demonstrated effectiveness, favorable safety profile, andbecause of disadvantages of systemic aminoglycosides and nonabsorbabledisaccharides. Rifaximin has a broad spectrum of in vitro antibacterialactivity against both Gram-positive and Gram-negative bacteria andagainst aerobic and anaerobic isolates.

Since rifaximin is poorly absorbed after oral administration, the drugis selectively active in the gastrointestinal tract. Additionally, thereis a low risk of drug-drug interactions with the use of rifaximin.Rifaximin has a lower rate of fecal eradication of pathogens comparedwith other commonly used antibacterial drugs and causes minimalalterations in gut flora suggesting that rifaximin has a differentmechanism of action than other commonly used drugs in enteric bacterialinfection, such as the fluoroquinolones. The risk of the development ofantibiotic resistance is low during chronic treatment with rifaximinwhen compared to other systemic antibiotics such as neomycin, possiblybecause resistance is mediated by a mutation in host cell DNA and is notplasmid based.

In a retrospective chart review, the numbers and durations ofhospitalizations due to HE, the total cost of therapy, and HE endpoints(asterixis grade, Conn score) were found to be dramatically reduced whencompared to lactulose treatment in patients with HE who receivedlactulose daily for 6 months and then received rifaximin daily for 6months.

The first study was designed to overcome the limitations of previousstudies reported in the literature (e.g., heterogeneous subjectpopulations, small population size, short durations, and insufficientendpoints for mental status).

First, treatment duration was increased to 6 months. This longerduration was planned to allow for a greater number of subjects toexperience an HE episode than if the study was limited to ≤6 weeks.Also, the longer treatment duration provided an opportunity to evaluatethe long-term safety of rifaximin in subjects with chronic hepaticcirrhosis and associated recurrent, overt, episodic HE. The studyinvestigated consequences of HE with respect to patient care andeconomic cost by measuring hospitalizations due to HE episodes as a keysecondary efficacy endpoint.

To evaluate overt HE episodes by using clinically relevant criteria inthe first study and study the second study, mental status impairment wasmeasured by using Conn score (West Haven criteria) and the severity ofneuromotor abnormalities was measured by asterixis grade. The Conn scoreranges from Stage 0 (lack of detectable changes in personality) to Stage4 (coma, decerebrate posturing, dilated pupils). The Conn score is therecommended and widely used gold standard for grading the severity ofimpaired mental status in overt HE. Asterixis (flapping tremor) is aneuromotor symptom of overt HE that increases in severity with worseningneurological impairment.

The control group for the first study received matched placebo tabletsin parallel with rifaximin treatments in the active group. The secondstudy was an ongoing open-label, treatment-extension study to evaluatethe long-term safety of rifaximin 550 mg BID in subjects with a historyof recurrent, episodic, overt HE. In addition to safety measurements,Conn scores and asterixis grades were assessed during the course of thestudy to measure the protective effect of rifaximin against breakthroughovert HE during treatment for up to approximately 1 year in subjects whocompleted up to 6 months of rifaximin treatment in the first study andthen entered the second study; in subjects who received placebo in thefirst study and crossed over to rifaximin treatment in the second study;and in patients with a history of HE who entered the second study as newsubjects.

The dosage regimen used (550 mg BID) was based on past clinicalexperience with rifaximin in patients with HE and other subjectpopulations. In several previous studies, rifaximin was safe andeffective in subjects with HE at a dose of 1200 mg per day with orwithout concomitant lactulose. In a 6-month study of rifaximin versusneomycin (14 days on-treatment and 14 days off-treatment per month),⁸rifaximin 1200 mg/day and neomycin (3 g/day) had comparable efficacy inpatients with HE. Aminoglycoside antibiotics are contraindicated inpatients with advanced liver disease because of the risk ofnephrotoxicity.

An efficacy endpoint was the time to first breakthrough overt HEepisode. A breakthrough overt HE episode was defined as an increase ofConn score to Grade≥2 (e.g., 0 or 1 to ≥2) or an increase in Conn andasterixis score of 1 grade each for those subjects who entered the studywith a Conn score of 0. Time to breakthrough overt HE episode was theduration from time of first dose of study drug to the first breakthroughovert HE episode. Subjects who completed the study and did notexperience a breakthrough overt HE episode were censored at the time oftheir 6-month visit. Subjects who terminated early for reasons otherthan breakthrough overt HE were contacted at 6 months from randomizationto determine if subjects had experienced a breakthrough overt HE episodeor other outcome (e.g., mortality status);

and, if the subject had no breakthrough overt HE event prior to contact,he/she was censored at the time of contact. Therefore, complete capturewas achieved for breakthrough overt HE episodes up to 6 monthspostrandomization. Subjects in the study had ≥2 episodes of overt HEequivalent to Conn score≥2 within 6 months prior to screening (i.e.,subjects had documented recurrent, overt HE). At the baselineassessment, subjects were in remission with a Conn score of 0 or 1. Abreakthrough overt HE episode, as defined above, was a markeddeterioration in neurological function.

Other efficacy endpoints in the first study included, for example:

1. Time to first HE-related hospitalization;2. Time to any increase from baseline in Conn score (mental stategrade);3. Time to any increase from baseline in asterixis grade;4. Mean change from baseline in fatigue domain scores on the CLDQ at endof treatment; and5. Mean change from baseline in venous ammonia concentration at end oftreatment.

Presented herein are the results of the first study and second study.The first study was a double-blind, randomized, placebo-controlled studyevaluating the efficacy and safety of rifaximin 550 mg BID as comparedto placebo. Subjects in remission from demonstrated recurrent, overt,episodic HE associated with chronic, hepatic cirrhosis were randomizedon Day 0 (Visit 2) according to a 1:1 ratio to receive rifaximin 550 mgBID or placebo for 6 months. The primary efficacy endpoint was the timeto breakthrough overt HE. Breakthrough overt HE was defined as anincrease of Conn score to Grade≥2 (e.g., 0 or 1 to ≥2) or an increase inConn and asterixis score of 1 grade each for those subjects who enteredthe study with a Conn score of 0. Subjects discontinued from the studyat the time of breakthrough overt HE episode. After participation in thefirst study, subjects had the option to enroll in the open-label,treatment-extension study (the second study).

A total of 299 subjects were randomized to receive rifaximin (140subjects) or placebo (159 subjects). All randomized subjects received atleast 1 dose of study drug. A total of 251 (84%) (116 [rifaximin], 135[placebo]) subjects completed the study as specified in the protocol(e.g., completed 6 months of treatment or withdrew from the study at thetime of breakthrough overt HE).

Subjects in the study had ≥2 episodes of overt HE equivalent to Connscore≥2 within 6 months prior to screening (e.g., subjects hadrecurrent, overt HE). At the baseline assessment, subjects were inremission with a Conn score of 0 or 1. A breakthrough overt HE episodewas a marked deterioration in neurological function. Breakthrough overtHE episodes were experienced by 31 of 140 subjects in the rifaximingroup and by 73 of 159 subjects in the placebo group during the 6-monthtreatment period (up to Day 170). Comparison of Kaplan-Meier estimatesof time to breakthrough overt HE between groups showed a protectiveeffect of rifaximin (p<0.0001). These data show that rifaximin treatmentresulted in a 57.9% reduction, when compared with placebo, in the riskof experiencing breakthrough overt HE. Rifaximin treatment results infewer overt HE episodes that may otherwise incapacitate the patient, mayalleviate the burden on family members who are required to care for thepatient, and reduces the burden of hospitalization in this patientpopulation and the healthcare system.

The following prognostic factors were found to be predictors ofbreakthrough overt HE episodes: baseline age (p=0.0160), MELD score(p=0.0003), duration of current verified remission (p=0.1089), andnumber of prior HE episodes (p=0.0022). These data show that rifaximintreatment, resulted in a 60% reduction, when compared with placebo, inthe risk of experiencing a breakthrough overt HE episode during thecourse of this study (p<0.0001).

Time to First HE-Related Hospitalization; and the Frequencies ofHE-Related and All-Cause Hospitalizations

Hepatic encephalopathy-related hospitalizations (hospitalizationdirectly resulting from HE or hospitalization complicated by HE) werereported for 19 of 140 subjects and 36 of 159 subjects in the rifaximinand placebo groups, respectively. Rifaximin had a protective effectagainst HE-related hospitalization during the 6-month treatment period.Subjects in the rifaximin group had a 50% reduction in the risk ofhospitalization due to HE during the 6-month treatment period whencompared with placebo. The HE-related hospitalization rate was 0.38events/PEY in the rifaximin group versus 0.78 event/PEY in the placebogroup.

Hepatic encephalopathy-caused hospitalizations (hospitalization directlyresulting from HE only) were reported for 15 of 140 subjects and 33 of159 subjects in the rifaximin and placebo groups, respectively.Rifaximin had a significant protective effect against HE-causedhospitalization during the 6-month treatment period; hazard ratio in therifaximin group relative to placebo was 0.438 (95% CI: 0.238 to 0.807)(p=0.0064) for the risk of HE-caused hospitalization. Subjects in therifaximin group had a 56% reduction in the risk of hospitalization dueto HE during the 6-month treatment period when compared with placebo.The HE-caused hospitalization rate was 0.30 events/PEY in the rifaximingroup versus 0.72 event/PEY in the placebo group.

All-cause hospitalization was also lower in the rifaximin group (46 of140) than in the placebo group (60 of 159) (30% reduction in therifaximin group compared with placebo). The all cause hospitalizationrate, after normalizing for subject exposure, was 0.90 events/PEY in therifaximin group and 1.26 event/PEY in the placebo group. The HE-relatedhospitalization rate was 0.38 event/PEY in the rifaximin group and 0.78event/PEY in the placebo group. Rifaximin treatment protects againstHE-related hospitalization, thereby improving the quality of life forthe patient and for his/her caregiver, and reducing the economic costrelated to liver cirrhosis and associated HE.

Time to Any Increase from Baseline in Conn Score and Time to AnyIncrease from Baseline in Asterixis Grade

Protective effects of rifaximin were observed with respect to both ofthese endpoints when analyzed independently; hazard ratio in therifaximin group relative to placebo was 0.463 (95% CI: 0.312 to 0.685)(p<0.0001) for the risk of experiencing an increase in Conn score and0.646 (95% CI: 0.414 to 1.008) (p=0.0523) for the risk of experiencingan increase in asterixis grade during the 6-month treatment period.

Changes From Baseline in Venous Ammonia Levels at End of Treatment

Subjects in the rifaximin group had greater reductions in venous ammonialevels when compared to placebo-treated subjects (p=0.0391). Venousammonia levels, a quantitative assessment that is associated with theCNS effects underlying overt HE, was found to be highly correlated tothe occurrence of breakthrough overt HE as determined by the clinicalevaluation of Conn score (or a combination of Conn score and asterixisgrade).

Tracking of Conn Scores and Asterixis Grades: Changes from Baseline inConn Scores and Asterixis Grades

A favorable treatment effect of rifaximin was observed, when comparedwith placebo, with respect to the proportions of subjects who hadchanges of -1 (improvement) or 0 (no change); or 1, 2, or 3 (worsening)in Conn score from baseline to end of treatment (last postbaselineassessment or assessment at time of breakthrough HE). In the rifaximingroup compared to placebo, higher proportions of subjects experiencedConn score changes of −1 or no change (77.1% versus 53.9%) and lowerproportions of subjects had Conn score changes of 1, 2, 3, or 4. Thus,treatment with rifaximin was more effective than placebo in theprevention of worsening of Conn score (2.46 times versus placebo,p=<0.0001).

For changes from baseline to end of treatment in asterixis grade,significantly higher proportions of subjects in the rifaximin groupversus the placebo group had changes from baseline in asterixis gradesof −2, −1, and 0 (88.5% versus 77.0%), and significantly lowerproportions of subjects had changes of 1, 2, 3, or 4 (11.6% versus23.2%). Thus, treatment with rifaximin was more effective than placeboin the prevention of worsening of asterixis grade (1.92 times versusplacebo, p=0.0262).

Changes from Baseline in CFF Results

Increases in CFF results represent improvement in neurological functionin patients with HE.^(10,11) Subjects in the rifaximin group hadsignificantly greater increases in CFF results from baseline to end oftreatment when compared with placebo. Mean changes (±standard deviation[SD]) in CFF results were 0.945 (±4.75) in the rifaximin group versus0.355 (±4.70) in the placebo group (p=0.0320 for between-groupdifference). Similar to venous ammonia levels, CFF was shown to behighly predictive of breakthrough HE.

Median exposure to study drug was 168 days (range: 10 to 178) in therifaximin group and 110 days (range: 6 to 176) in the placebo group. Atotal of 64 subjects (33 [rifaximin] and 31 [placebo]) receivedtreatment for 141 to 168 days and 98 subjects (57 [rifaximin] and 41[placebo]) received treatment for >168 days. Duration of exposureresults are consistent with the finding that lower proportions ofsubjects in the rifaximin group than in the placebo group experiencedbreakthrough overt HE resulting in study discontinuation (per protocol,subjects discontinued from the study after breakthrough overt HE).

The percentages of subjects who had treatment-emergent AEs, severeTEAEs, drug-related TEAEs, treatment-emergent SAEs, TEAEs resultingdiscontinuation, and who died were similar between placebo and rifaximingroups. A total of 79.9% of subjects (239 of 299) experienced TEAEsduring the course of the study. The most common TEAEs (e.g., in >10% oftotal subjects [combined placebo plus rifaximin]) experienced bysubjects were the following: diarrhea (10.7% [rifaximin] versus 13.2%[placebo]), nausea (14.3% versus 13.2%), peripheral edema (15% versus8.2%), fatigue (12.1% versus 11.3%), dizziness (12.9% versus 8.2%),ascites (11.4% versus 9.4%), and headache (10% versus 10.7%).

The second study is an ongoing open-label, treatment-extension studyevaluating the long-term safety of rifaximin 550 mg BID in subjects witha history of recurrent, overt, episodic HE. All eligible subjects had ahistory of overt HE episodes with a documented severity equivalent toConn score≥2 within 12 months prior to screening (≥1 qualifying episodewas required), a Conn score of ≤2 at the baseline assessment, and eitherparticipated in the first study or were new subjects. Unlike the firststudy, subjects were not required to withdraw from the study afterexperiencing a breakthrough overt HE episode.

A total of 267 subjects were enrolled and 208 were active at the time ofthe interim clinical cutoff. Additional data were collected for theinterim report up to the time of database freeze.

Conn scores and asterixis grades were assessed during the course of thestudy. Therefore, it was possible to determine time to breakthroughovert HE episode for subjects who completed 6 months of rifaximintreatment in the first study and then entered the second study, subjectswho received placebo in the first study and then started rifaximin inthe second study, and in new subjects who started rifaximin therapy inthe second study. In subjects who took rifaximin for up to 680 days (1.9years), breakthrough overt HE episodes during the treatment period wereexperienced by 72 of 266 subjects (27.1%) overall: 54 of 196 subjects(27.6%) in the new rifaximin group and 18 of 70 subjects (25.7%) in thecontinuing rifaximin group.

Time-to-first-breakthrough HE profiles were similar between therifaximin group in the first study and the new rifaximin group in thesecond study. A durable protective effect of rifaximin was observed insubjects who received rifaximin starting in the first study andcontinuing in the second study (median exposures to rifaximin were 168days in the first study and 253 days in the second study)

A total of 133 of 266 subjects were hospitalized for any cause: 98 inthe new rifaximin group, and 35 in the continuing rifaximin group.Normalizing for subject exposure, this represents a hospitalization rateof 0.60 event/PEY. A total of 59 were hospitalized due HE episodes(e.g., HE-caused). Normalizing for subject exposure, this represents anHE-caused hospitalization rate of 0.29 event/PEY. The low HE-causedhospitalization rate was consistent between rifaximin therapy in thesecond study (0.29 event/PEY) and in the first study rifaximin (0.30event/PEY) at least partly as a result of maintaining remission fromdemonstrated HE in subjects with end-stage liver disease. Tracking ofConn scores and asterixis grades: changes from baseline in Conn scoresand asterixis grades Conn scores were generally maintained or improvedwith rifaximin use up to 18 months. At the last visit, 70.7% of subjects(188 of 266 subjects) had no change and 20.3% (54 of 266) hadimprovements in Conn scores compared with baseline, indicating thatmental status was maintained or improved in the majority of subjects(91%) over the treatment period. Of the 84 subjects (70 new rifaximinand 14 continuing rifaximin) who entered the study with Conn scores of1, 2, or 3 (e.g., those subjects for whom measurable improvement waspossible), 54 subjects (54/84=64.3%) showed a 1-grade (47 subjects;56.0%) or 2-grade (7 subjects; 8.3%) improvement from baseline at thelast visit recorded for the interim analysis. All subjects were capableof worsening over time, and 24/266 subjects (9.0%) did so by 1 or 2grades.

Like Conn scores, asterixis grades were generally maintained or improvedwith rifaximin use up to 18 months. At the last visit, 77.1% of subjects(205 of 266 subjects) had no change and 16.2% (43 of 266) hadimprovements in asterixis scores compared with baseline, indicating thatneuromotor symptoms associated with increasing neurological impairmentwere maintained in 83.3% of subjects over the treatment period. Of the67 subjects (55 new rifaximin and 12 continuing rifaximin) who enteredthe study with asterixis scores of 1, 2, or 3 (e.g., those subjects forwhom improvement was possible), 43 subjects (43/67=64.2%) showed a 1-(34 subjects; 50.7%), 2- (4 subjects; 6.0%), or 3-grade (5 subjects;7.5%) improvement from baseline at the last visit recorded for thisinterim analysis. All subjects were capable of worsening over time, and18/266 subjects (6.8%) did so by 1, 2, or 4 grades; the incidence ofworsening asterixis grades were similar between the new (12/196subjects; 6.1%) and continuing (6/70 subjects; 8.6%) rifaximin groups.

Median exposures in study the second study were 253 days (range: 7 to680) in the new rifaximin group (subjects who received placebo in thefirst study or subjects who did not participate in the first study),265.5 days (range: 10 to 673) in the continuing rifaximin group(subjects who received rifaximin in the first study and the secondstudy), and 255 days (range: 7 to 680) in the all rifaximin group (allsubjects who received rifaximin in the second study). At the time ofthis interim analysis, most subjects had received rifaximin for 6 to <9months (21.4%) or 9 to <12 months (32.3%).

At the time of this interim analysis, TEAEs were reported in 230subjects (86.5%). The most common TEAEs (e.g., in ≥10% of totalsubjects) experienced by subjects were the following: peripheral edema(15.8%); urinary tract infection and nausea (12.8% each); and abdominalpain and ascites (10.5% each). Note that signs and symptoms associatedwith HE were not considered AEs unless they met the definition of anSAE, so the number of subject with HE counted in efficacy analysis (72subjects; 27.1%) is higher than that counted for the safety analyses (57subjects; 21.4%).

Most TEAEs were mild or moderate in intensity, with 40.2% of subjectsexperiencing at least 1 TEAE that was judged by the investigator to besevere. The incidence of TEAEs considered related to study drug wascomparable between the new rifaximin group (7.7%) and the continuingrifaximin group (7.1%). Treatment-emergent SAEs were experienced by47.4% of subjects.

FIG. 1 illustrates Kaplan-Meier estimates of time to first breakthroughovert HE episode by treatment group in the ITT population. Table 18presents Kaplan-Meier estimates of the proportions of subjects whoexperienced breakthrough overt HE over the course of the TreatmentPeriod and results of statistical analyses. Subjects who completed thestudy and did not experience a breakthrough overt HE event were censoredat the time of their 6-month visit. Subjects who terminated early forreasons other than breakthrough overt HE (eg, liver transplant, AE,subject request) were contacted at 6 months from date of randomizationto determine if subjects had experienced a breakthrough overt HE episodeor other outcome (e.g., mortality status). Subjects without breakthroughovert HE were censored at the time of contact or death, whichever wasearlier. Therefore, complete capture was achieved for breakthrough overtHE episodes up to 6 months.

TABLE 18 The First Study: Kaplan-Meier Estimates and StatisticalAnalyses of Time to First Breakthrough Overt HE (up to 6 Months ofTreatment, Day 170) (ITT Population) Placebo (N = 159) Rifaximin (N =140) Probability Probability Treatment Number Cumulative Event of noNumber Cumulative Event of no interval At of number probabilitybreakthrough At of number probability breakthrough (days) Risk^(a)events^(b) of events (SE)^(c) overt HE^(d) Risk^(a) events^(b) of events(SE)^(c) overt HE^(d) 0 to 158 20 20 0.13 1.0000 140 13 13 0.09 1.0000<28 (0.03) (0.02) 28 to 137 23 43 0.17 0.8734 126 4 17 0.03 0.9071 <56(0.03) (0.02) 56 to 113 14 57 0.12 0.7262 120 6 23 0.05 0.8783 <84(0.03) (0.02) 84 to 98 10 67 0.10 0.6363 112 7 30 0.06 0.8344 <140(0.03) (0.02) 140 to 84 6 73 0.07 0.5713 98 1 31 0.01 0.7820 <168 (0.03)(0.01) ≥168 38 0 73 0 0.5305 46 0 31 0 0.7740 Hazard ratio: 0.421^(e)95% CI: (0.276, 0.641) p-value < 0.0001 ^(a)Number of subjects at riskduring the treatment interval, estimated using the life table method.Assuming that censored cases were at risk for half of the interval, theyonly counted for half in figuring the number at risk. ^(b)Number ofevents occurring during the treatment interval. ^(c)Estimate of theprobability of experiencing breakthrough overt HE during the treatmentinterval. Standard error (SE) estimated by using Greenwood's formula.^(d)Estimate of the probability of no breakthrough overt HE until atleast the beginning of the next treatment interval. ^(e)Hazard ratioestimate (hazard of breakthrough overt HE in the rifaximin groupcompared with the placebo group) determined from the Cox proportionalhazards model. P-value based on the Score statistic.

Breakthrough overt HE episodes were experienced by 31 of 140 subjects inthe rifaximin group and by 73 of 159 subjects in the placebo groupduring the 6-month period since randomization (up to Day 170).Comparison of Kaplan-Meier estimates of time to breakthrough overt HEbetween groups showed a protective effect of rifaximin (p<0.0001). Thesedata show that rifaximin treatment resulted in a 57.9% reduction, whencompared with placebo, in the risk of experiencing breakthrough overt HEduring the course of this study. Rifaximin treatment results in fewerovert HE episodes that may otherwise incapacitate the patient, mayalleviate the burden on family members who are required to care for thepatient, and reduces the burden of hospitalization in this patientpopulation and the healthcare system.

To investigate the potential effect of prognostic factors onbreakthrough overt HE episode, the following prognostic factors wereexamined:

Sex (male vs. female);

Age;

Race (white vs. non-white);Analysis Region (North American vs. Russia);

MELD Level; Conn Score (0 vs. 1);

Diabetes at Baseline (Yes vs. No);Duration of current verified remission; andNumber of HE Episodes within the past 6 months prior to randomization.

Strong independent predictors of breakthrough overt HE episodes were thebaseline age (p=0.0160), MELD score (p=0.0003), duration of currentverified remission (p=0.1089), and number of prior HE episodes(p=0.0022).

These data show that rifaximin treatment, after adjusting forsignificant prognostic factors, resulted in a 60% reduction, whencompared with placebo, in the risk of experiencing a breakthrough overtHE episode during the course of this study. The most influentialprognostic factors were age (p=0.0315) and baseline MELD score(p=0.0003).

The results indicate that the highly significant protective effect ofrifaximin (p<0.0001) against breakthrough overt HE episodes wasmaintained in the presence of statistically significant competingfactors.

In the second study, median exposures were 253 days (range: 7 to 680) inthe new rifaximin group (subjects who received placebo in the firststudy or subjects who did not participate in the first study), 265.5days (range: 10 to 673) in the continuing rifaximin group (subjects whoreceived rifaximin in the first study and the second study), and 255days (range: 7 to 680) in the all rifaximin group (all subjects whoreceived rifaximin in the second study

In subjects who took rifaximin for up to 680 days (1.9 years),breakthrough overt HE episodes during the treatment period wereexperienced by 72 of 266 subjects (27.1%) overall: 54 of 196 subjects(27.6%) in the new rifaximin group and 18 of 70 subjects (25.7%) in thecontinuing rifaximin group. FIG. 2 compares subjects who participated inthe double-blind, randomized the first study with new rifaximin subjectsin the long-term, open-label study, the second study.

The Kaplan-Meier estimates of time to first breakthrough overt HEepisode were similar between the rifaximin group in the first study andnew rifaximin subjects in the second study. Also, similar proportions ofsubjects had breakthrough overt HE in the rifaximin group of the firststudy (22%, 31 of 140 [rifaximin group]) and in the new rifaximin groupof the second study (27.6%, 54 of 196). Adjusted for exposure, rates ofbreakthrough HE episodes were 0.62 events/PEY in the rifaximin groupfrom the first study compared to 0.38 events/PEY for new rifaximinsubjects in the second study. These data demonstrate that protectionagainst breakthrough overt HE in subjects who received rifaximin wasconsistent between the 2 studies.

Note for FIG. 7 , the survival distribution estimate on y-axisrepresents the proportion of subjects without breakthrough overt HE.

The first study data on time to first breakthrough overt HE episode areshown for the rifaximin group (small dashes) and the placebo group(straight line). The second study data on time to first breakthroughovert HE episode in the new rifaximin group are shown in large dashes.

In FIG. 8 , the first study placebo subjects were followed after theycrossed over to rifaximin therapy in the second study. Breakthroughovert HE was experienced by 15 of 82 during rifaximin treatment versus39 of 82 during placebo treatment. A striking protective effect ofrifaximin was observed in the comparison of Kaplan-Meier estimates oftime to first breakthrough overt HE between placebo experience in thefirst study and rifaximin experience in the second study. The hazardratio of rifaximin to placebo was 0.302 (95% CI: 0.166 to 0.549,p<0.0001 for between group difference in relative risk). This resultrepresents 70% reduction in risk of experiencing breakthrough overt HEduring rifaximin treatment in the second study when compared with theirprior placebo experience in the first study.

Note for FIG. 8 , the survival distribution estimate on y-axisrepresents the proportion of subjects without breakthrough overt HE. thefirst study data on time to first breakthrough overt HE episode areshown in the left panel for the placebo group. The right panel showstime to first breakthrough overt HE in the second study among the firststudy placebo subjects (n=82) who crossed over to rifaximin therapy inthe second study. The vertical line between the left and right panelsmarks the end of the double-blind study and start of the open-labelstudy.

FIG. 9 illustrates time to first HE-related hospitalization (e.g.,hospitalization directly resulting from HE or hospitalization caused byHE) by treatment group in the ITT population in the first study. Table19 presents estimates of the proportions of subjects who had their firstHE-related hospitalization over the course of the Treatment Period andresults of statistical analyses. Subjects who discontinued prior tohospitalization due to HE and prior to completion of the 6-monthtreatment period were censored at the time of discontinuation. Hepaticencephalopathy-related hospitalizations were reported for 19 of 140subjects and 36 of 159 subjects in the rifaximin and placebo groups,respectively. Rifaximin had a protective effect against HE-relatedhospitalization during the 6-month treatment period; hazard ratio in therifaximin group relative to placebo was 0.500 (95% CI: 0.287 to 0.873)(p=0.0129) for the risk of HE-related hospitalization. This hazard ratiorepresents a 50% reduction, when compared with placebo, in the risk ofhospitalization due to HE during the 6-month treatment period.Consistent with these results, the HE-related hospitalization rate was51% lower (0.38 event/PEY, rifaximin versus 0.78 event/PEY, placebo) inthe rifaximin group in the first study, after normalization to exposure.

Note for FIG. 9 , the survival distribution estimate on y-axisrepresents the proportion of subjects without HE-relatedhospitalization. Dashed line represents rifaximin group and solid linerepresents placebo group. Open circles and open triangles representcensored subjects. Subjects who discontinued prior to hospitalizationdue to HE and prior to completion of the 6-month treatment period werecensored at the time of discontinuation. Hepatic encephalopathy-relatedhospitalization was recorded on the HE-related hospitalization CRF.

TABLE 19 The First Study: Kaplan-Meier Estimates and StatisticalAnalyses of Time to First HE-Related Hospitalization (up to 6 Months ofTreatment, Day 170) (ITT Population) Placebo (N = 159) Rifaximin (N =140) Probability Probability Treatment Number Cumulative Event of noNumber Cumulative Event of no interval At of number probabilityHE-related At of number probability HE-related (days) Risk^(a)events^(b) of events (SE)^(c) hospitalization^(d) Risk^(a) events^(b) ofevents (SE)^(c) hospitalization^(d) 0 to 155 11 11 0.07 1.0000 139 4 40.03 1.0000 <28 (0.02) (0.01) 28 to 132 12 23 0.09 0.9288 130 4 8 0.030.9711 <56 (0.03) (0.02) 56 to 108 7 30 0.06 0.8440 119 4 12 0.03 0.9411<84 (0.02) (0.02) 84 to 88 4 34 0.05 0.7893 106 5 17 0.05 0.0904 <140(0.02) (0.02) 140 to 72 2 36 0.03 0.7535 92 2 19 0.02 0.8665 <168 (0.02)(0.02) ≥168 34 0 36 0 0.7525 43 0 19 0 0.8475 Abbreviations: CI =confidence interval; SE = standard error. ^(a)Number of subjects at riskduring the treatment interval, estimated using the life table method.Assuming that censored cases were at risk for half of the interval, theyonly counted for half in figuring the number at risk. ^(b)Number ofevents occurring during the treatment interval. ^(c)Estimate of theprobability of experiencing HE-related hospitalization during thetreatment interval. Standard error (SE) estimated by using Greenwood'sformula. ^(d)Estimate of the probability of no HE-relatedhospitalization until at least the beginning of the next treatmentinterval. ^(e)Hazard ratio estimate (hazard of HE-relatedhospitalization in the rifaximin group compared with the placebo group)determined from the Cox proportional hazards model. P-value based on theScore statistic.

The effect of rifaximin therapy on HE-caused hospitalizations (e.g.,hospitalization directly resulting from HE only) was also determined.FIG. 5 illustrates time to first HE-caused hospitalizations by treatmentgroup in the first study.

Hepatic encephalopathy-caused hospitalizations were reported for 15 of140 subjects and 33 of 159 subjects in the rifaximin and placebo groups,respectively. Rifaximin had a significant protective effect againstHE-caused hospitalization during the 6-month treatment period; hazardratio in the rifaximin group relative to placebo was 0.438 (95% CI:0.238 to 0.807) (p=0.0064) for the risk of HE-caused hospitalization.Subjects in the rifaximin group had a 56% reduction in the risk ofhospitalization due to HE during the 6-month treatment period whencompared with placebo. The HE-caused hospitalization rate was 0.30events/PEY in the rifaximin group versus 0.72 event/PEY in the placebogroup.

Note for FIG. 10 , the survival distribution estimate on y-axisrepresents the proportion of subjects without HE-causedhospitalizations. Dashed line represents rifaximin group and solid linerepresents placebo group. Open circles and open triangles representcensored subjects. Subjects who discontinued prior to hospitalizationwere censored at the time of discontinuation.

The effect of rifaximin therapy on all-cause hospitalizations was alsodetermined. In the double-blind the first study, 46 of 140 rifaximinsubjects and 60 of 159 placebo subjects were hospitalized due to anySAE. The risk of all-cause hospitalization was reduced by 30% in therifaximin group when compared to placebo (p=0.0793 for between-groupdifference in relative risk). The all-cause hospitalization rate was0.92 events/PEY in the rifaximin group versus 1.31 event/PEY in theplacebo group. These data demonstrated that rifaximin treatment reducedthe burden of HE-related/caused hospitalization when compared to placebotreatment in the first study. Also, a low HE-related/causedhospitalization rate was consistently observed during rifaximin therapyin the first study (0.38 event/PEY) and in the second study (0.29event/PEY), at least partly as a result of maintaining remission fromdemonstrated HE in subjects with end-stage liver disease.

FIG. 11 illustrates time to any increase from baseline in Conn score bytreatment group in the ITT population. Table 20 presents estimates ofthe proportions of subjects who had any increase in Conn score over thecourse of the Treatment Period and results of statistical analyses.Subjects who discontinued prior to experiencing an increase in Connscore and prior to completion of the 6-month treatment period werecensored at the time of discontinuation. By evaluating the time to anyincrease from baseline in Conn score, it was possible to compare theearliest worsening in mental status between subjects in the rifaximinand placebo treatment groups, even if the worsening did not reach thedefinition of breakthrough HE (eg, increase inConn score from 0 to 1).Increases in Conn score were reported for 37 of 140 subjects and 77 of159 subjects in the rifaximin and placebo groups, respectively. A highlysignificant protective effect of rifaximin was observed; hazard ratio inthe rifaximin group relative to placebo was 0.463 (95% CI: 0.312 to0.685) (p<0.0001) for the risk of experiencing an increase in Conn score(e.g., worsening in mental status) during the 6-month treatment period.

TABLE 20 The First Study: Kaplan-Meier Estimates and StatisticalAnalyses of Time to First Increase in Conn Score (up to 6 Months ofTreatment, Day 170) (ITT Population) Placebo (N = 159) Rifaximin (N =140) Probability Probability Treatment Number Cumulative Event of noNumber Cumulative Event of no interval At of number probability increasein At of number probability increase in (days) Risk^(a) events^(b) ofevents (SE)^(c) Conn score^(d) Risk^(a) events^(b) of events (SE)^(c)Conn score^(d) 0 to 156 26 26 0.17 1.0000 139 17 17 0.012 1.0000 <28(0.03) (0.03) 28 to 125 21 47 0.17 0.8333 119 5 22 0.04 0.8777 <56(0.03) (0.02) 56 to 100 15 62 0.15 0.6928 109 9 31 0.08 0.8407 <84(0.04) (0.03) 84 to 80 10 72 0.13 0.5883 94 5 36 0.05 0.7713 <140 (0.04)(0.02) 140 to 62 5 77 0.08 0.5143 79 0 36 0 0.7302 <168 (0.03) ≥168 27 077 0 0.4729 37 1 37 0.03 0.7302 (0.03) Abbreviations: CI = confidenceinterval; SE = standard error. ^(a)Number of subjects at risk during thetreatment interval, estimated using the life table method. ^(b)Number ofevents occurring during the treatment interval. Assuming that censoredcases were at risk for half of the interval, they only counted for halfin figuring the number at risk. ^(c)Kaplan-Meier estimate of theprobability of experiencing an increase in Conn score during thetreatment interval. Standard error (SE) estimated by using Greenwood'sformula. ^(d)Estimate of the probability of no increase in Conn scoreuntil at least the beginning of the next treatment interval. ^(e)Hazardratio estimate (hazard of experiencing an increase in Conn score in therifaximin group compared with the placebo group) determined from the Coxproportional hazards model. P-value based on the Score statistic.

FIG. 12 illustrates time to any increase from baseline in asterixisgrade by treatment group in the ITT population in the first study. Table21 presents estimates of the proportions of subjects who had anyincrease in asterixis grade over the course of the Treatment Period andresults of statistical analyses. Subjects who discontinued prior toexperiencing an increase in asterixis grade and prior to completion ofthe 6-month treatment period were censored at the time ofdiscontinuation.

By evaluating the time to any increase from baseline in asterixis grade,it was possible to compare the earliest worsening in neuromotorfunctioning between subjects in the rifaximin and placebo treatmentgroups. Increases in asterixis grade were reported for 32 of 140subjects and 50 of 159 subjects in the rifaximin and placebo groups,respectively. A protective effect of rifaximin against an increase inasterixis grade (e.g., worsening in neuromotor functioning) was observedthat showed a trend toward statistical significance; hazard ratio in therifaximin group relative to placebo was 0.646 (95% CI: 0.414 to 1.008)(p=0.0523) for the risk of experiencing an increase in asterixis gradeduring the 6-month treatment period.

TABLE 21 The First Study: Kaplan-Meier Estimates and StatisticalAnalyses of Time to First Increase in Asterixis Grade (up to 6 Months ofTreatment, Day 170) (ITT Population) Placebo (N = 159) Rifaximin (N =140) Probability Probability of no of no Treatment Number CumulativeEvent increase in Number Cumulative Event increase in interval At ofnumber probability asterixis At of number probability asterixis (days)Risk^(a) events^(b) of events (SE)^(c) grade^(d) Risk^(a) events^(b) ofevents (SE)^(c) grade^(d) 0 to 154 20 20 0.13 1.0000 137 13 13 0.101.0000 <28 (0.03) (0.03) 28 to 120 15 35 0.13 0.8697 116 7 20 0.060.9048 <56 (0.03) (0.02) 56 to 91 4 39 0.04 0.7610 101 7 27 0.07 0.8499<84 (0.02) (0.03) 84 to 76 6 45 0.08 0.7275 87 3 30 0.03 0.7910 <140(0.03) (0.02) 140 to 61 4 49 0.07 0.6701 74 1 31 0.01 0.7637 <168 (0.03)(0.01) ≥168 27 1 50 0.04 0.6262 34 1 32 0.03 0.7534 (0.04) (0.03)Abbreviations: CI = confidence interval; SE = standard error. ^(a)Numberof subjects at risk during the treatment interval, estimated using thelife table method. Assuming that censored cases were at risk for half ofthe interval, they only counted for half in figuring the number at risk.^(b)Number of events occurring during the treatment interval.^(c)Estimate of the probability of experiencing an increase in asterixisgrade during the treatment interval. Standard error (SE) estimated byusing Greenwood's formula. ^(d)Estimate of the probability of noincrease in asterixis grade until at least the beginning of the nexttreatment interval. ^(e)Hazard ratio estimate (hazard of experiencing anincrease in asterixis grade in the rifaximin group compared with theplacebo group) determined from the Cox proportional hazards model.P-value based on the Score statistic.

Subjects ranked their level of fatigue by using a 7-point scale from theworst response (1, high degree of fatigue) the best response (7, minimalfatigue). Minimal differences between placebo and rifaximin groups wereobserved in the changes from baseline in CLDQ fatigue scores. Mean (SD)fatigue scores were 3.34 (1.406) versus 3.28 (1.326) at baseline and3.51 (1.529) versus 3.57 (1.527) in the placebo and rifaximin groups,respectively. Because of altered mental and neuromotor status, it wasnot possible for subjects to complete the CLDQ assessment during anovert HE breakthrough episode.

Table 22 summarizes changes from baseline to end of treatment in venousammonia level by treatment group in the first study.

In the first study, venous ammonia levels were highly variable over thecourse of the study. However, subjects in the rifaximin group hadsignificantly greater reductions in venous ammonia levels when comparedto placebo-treated subjects (p=0.0391). Venous ammonia levels, aquantitative assessment that is associated with the CNS effectsunderlying overt HE, was shown to be highly predictive of the occurrenceof breakthrough overt HE as determined by the clinical evaluation ofConn score (or a combination of Conn score and asterixis grade), therebyunderscoring the reliability and clinical relevance of the primaryefficacy measure. The significant correlation of the primary efficacyendpoint to a venous ammonia levels demonstrates the reliability andclinical relevance of the primary efficacy measure in the first study.

TABLE 22 The First Study: Mean (SD) Changes from Baseline in VenousAmmonia Level by Treatment Group (ITT Population) Placebo Placebo N =159 N = 140 (μg/dL) (μg/dL) Baseline n = 146 n = 132 Mean (SD) ammonialevel 90.3 (52.48) 87.9 (47.76) End of treatment n = 141 n = 132 Mean(SD) ammonia level 88.4 (45.75) 83.9 (45.02) Change from baseline to endof treatment n = 131 n = 125 Mean (SD) change in ammonia level −0.3(58.13) −5.7 (46.77)

Note: Baseline value was the last available value prior to first dose ofstudy drug, and end of treatment value was the last availablepost-baseline value during the treatment period.

The Second Study

In the second study, Conn scores were generally maintained or improvedwith rifaximin use up to 18 months. At the last visit, 70.7% of subjects(188 of 266 subjects) had no change and 20.3% (54 of 266) hadimprovements in Conn scores compared with baseline, indicating thatmental status was maintained or improved in the majority of subjects(91%) over the treatment period. Like Conn scores, asterixis grades weregenerally maintained or improved with rifaximin use up to 18 months. Atthe last visit, 77.1% of subjects (205 of 266 subjects) had no changeand 16.2% (43 of 266) had improvements in asterixis scores compared withbaseline, indicating that neuromotor symptoms associated with increasingneurological impairment were maintained in 83.3% of subjects over thetreatment period. The last visit for the second study is the last visitrecorded for the interim analysis.

Maintenance or improvement in Conn scores were observed for >85% ofsubjects during rifaximin treatment for up to 840 days; mean (±SD)exposure for all rifaximin experience was 273.8 (160.92) days (exposureresults are present in detail in the ISS, Module 5.3.5.3.2). A total of65.5% of subjects (220 of 337) had no change in Conn score and 21.1% (71of 337) had improvements in Conn score from baseline to last visit.Similarly, maintenance or improvements in asterixis grades were observedfor >90% of subjects during rifaximin treatment. No change from baselinein asterixis grade was reported for 75.2% of subjects (252 of 337), and17.3% had improvements.

Of the 118 subjects who entered the study with a Conn score of ≥1, e.g.,those subjects for whom improvement was possible, 62.2% (71 of 118)showed an improvement from baseline to Conn score 0 at last assessment.Also, of the 99 subjects who entered with an asterixis grade of ≥1, iethose subjects for whom improvement in asterixis grade was possible,58.6% (58 of 99) showed improvement in asterixis grade from baseline toend of study.

Changes from baseline in Conn scores and asterixis grades to last visitwere similar among new rifaximin subjects in the second study (e.g.,started rifaximin in 3002), continuing rifaximin subjects (e.g.,received rifaximin in the first study and in the second study), and allrifaximin experience subjects (e.g., received rifaximin in the firststudy or in the second study).

These results support those from the first study, in which treatmentwith rifaximin was significantly more effective than placebo in theprevention of worsening of Conn score (2.46 times versus placebo,p<0.0001) and in the prevention of worsening of asterixis grade (1.92times versus placebo, p=0.0262).

Changes from Baseline in CFF Results (The First Study)

Increases in CFF results represent improvement in neurological functionin patients with HE. Subjects in the rifaximin group had significantlygreater increases in CFF results from baseline to end of treatment whencompared with placebo (Table 23). Mean changes (±SD) in CFF results were0.945 (±4.75) in the rifaximin group versus 0.355 (±4.70) in the placebogroup (p=0.0320 for between-group difference).

Similar to the correlation for venous ammonia levels, there was a strongcorrelation between the quantitative assessment of CFF results and theoccurrence of breakthrough overt HE.

TABLE 23 Mean (SD) Changes from Baseline in CFF Test Results byTreatment Group (ITT Population) Placebo Rifaximin N = 159 N = 140 (Hz)(Hz) Baseline n = 159 n = 140 Mean (SD) CFF result 37.41 (6.03) 36.90(5.47) End of treatment n = 155 n = 139 Mean (SD) CFF result 37.60(5.98) 37.81 (4.88) Change from baseline to end of treatment n = 155 n =139 Mean (SD) change in CFF result 0.355 (4.70) 0.945 (4.75) Note:Baseline value was the last available value prior to first dose of studydrug, and end of treatment value was the last available post-baselinevalue during the treatment period.

A retrospective chart review was performed for 145 patients with HE whoreceived lactulose 30 mL twice daily for ≥6 months followed by treatmentwith rifaximin 400 mg 3 times/day for ≥6 months. Dramatic differenceswere observed in favor of rifaximin treatment. Compliance of ≥75% wassignificantly better during rifaximin treatment than during lactulosetreatment; 92% versus 31% of patients received ≥75% of scheduledrifaximin and lactulose doses, respectively. Total number ofhospitalizations, duration of hospitalizations, HE endpoints, and costof therapy were compared between the 2 treatment regimens. Significantlyfewer hospitalizations (0.5 versus 1.6) and days hospitalized (2.5versus 7.3 days) were reported for rifaximin treatment versus lactulosetreatment (p<0.001), and hospitalization charges per patient were$14,222 compared with $56,635 during rifaximin and lactulose treatments,respectively.

With respect to HE endpoints at the end of the treatment periods,asterixis was reported for 63% (rifaximin) versus 93% (lactulose) ofpatients (p<0.001), and Conn scores of 3 or 4 were observed for 6%(rifaximin) versus 25% (lactulose) (p<0.001). In addition, significantlymore patients had diarrhea, flatulence, and abdominal pain duringlactulose therapy than during rifaximin therapy (p<0.001).

Hospitalizations and cost of therapy were analyzed in a chart review of39 liver transplant patients who presented with HE Conn scores of 2during the interval from January 2004 to November 2005. Twenty-fourpatients were treated with lactulose and 15 were treated with rifaximin.Nineteen hospitalizations were reported for the lactulose group and 3hospitalizations for the rifaximin group. The average length of stay wassignificantly shorter in the rifaximin group than in the lactulose group(3.5 days [range, 3-4] versus 5.0 days [range, 3 to 10] [p<0.001]). Theaverage annual total cost of treatment (hospitalization, emergency roomvisit, and drug cost) per patient was $7958 for the rifaximin group and$13,285 for the lactulose group. Although the cost of rifaximin wassubstantially higher than the cost of lactulose, total cost of treatment(hospitalization plus drug cost) was 1.67-fold higher in patients whowere treated with lactulose.

Durability of Rifaximin Treatment Effect

Data from the second study provide information on the long-termdurability of rifaximin for the protection against breakthrough overt HEepisodes. Rifaximin treated subjects from the first study who were inremission at the end of the first study (6 months treatment) werefollowed during open-label study the second study (n=60). Time to firstbreakthrough HE episode is shown for the rifaximin rollover subjects(the first study plus the second study) and the first study placebosubjects in FIG. 15 . The incidence of breakthrough overt HE in theserollover rifaximin subjects was compared to placebo subjects in thefirst study . The incidence of breakthrough HE episode for rifaximinsubjects was dramatically lower than the first study placebo group(ratio of rollover rifaximin to placebo was 0.0797 after adjusting forexposure time, p<0.0001 for difference between rifaximin and placebo.

These results demonstrated that rifaximin had a durable protectiveeffect beginning in the first study and continuing in the second study(median exposures to rifaximin were 168 days in the first study and 253days in the second study).

Note for FIG. 13 , the survival distribution estimate on y-axisrepresents the proportion of subjects without breakthrough overt HE.Dashed lines represents rifaximin treated subjects from the first studywho were in remission at the end of the first study (6 months treatment)and were followed during open-label study the second study (n=60), andsolid line represents the placebo group in the first study . Thevertical line marks the end of the double-blind study and start of theopen-label study. Open circles represent censored subjects in the firststudy placebo group and open triangles represent censored subjects inthe continuing rifaximin group. Subjects who discontinued prior to thefirst breakthrough overt HE episode were censored at the time ofdiscontinuation.

Unlike the first study, in which subjects were discontinued from thestudy after experiencing their first breakthrough overt HE episode,subjects had the option of continuing rifaximin therapy in the secondstudy after experiencing breakthrough overt HE. Therefore, the incidenceof breakthrough overt HE over time during rifaximin therapy wasevaluated. Table 24 presents breakthrough overt HE episodes by totalnumber of HE episodes during the course of the study.

In the all rifaximin group, 27.1% of subjects (72 of 266) had ≥1breakthrough overt HE episode. Of the 72 subjects with breakthrough HE,most had 1 (44 subjects) or 2 (18 subjects) episodes. Ten subjects had 3or more breakthrough HE episodes in the second study.

TABLE 24 the second study: Breakthrough Overt HE Episodes by Number ofRepeat Episodes New Continuing All Rifaximin Rifaximin Rifaximin N = 196N = 70 N = 266 n (%) n (%) n (%) Subjects with ≥1 breakthrough 54 (27.6)18 (25.7) 72 (27.1) overt HE episode Total number of HE episodes^(a)during the study: 1 34 (17.3) 10 (14.3) 44 (16.5) 2 12 (6.1)  6 (8.6) 18(6.8)  3 4 (2.0) 0 4 (1.5) 4 1 (0.5) 1 (1.4) 2 (0.8) 5 1 (0.5) 0 1 (0.4)6 0 1 (1.4) 1 (0.4) 10  2 (1.0) 0 2 (0.8) Abbreviation: HE = hepaticencephalopathy ^(a)Number of HE episodes. Subjects were counted onlyonce for each number of overt HE episodes. For example, if a subjectexperienced 3 episodes, he/she was included in the row showing 3episodes only, and was not also counted in the rows for 2 and 1episodes.

Effect of Rifaximin on the Incidence of Overt HE Episodes (HE Burden)

The effect of rifaximin therapy on the incidence of overt HE episodes(e.g., burden of HE), the numbers of HE episodes in the first study orthe second study were compared to the numbers of HE episodes in theabsence of rifaximin therapy. The 6-month interval prior to the firststudy or the 12-month interval prior to the second study was comparedagainst rifaximin therapy in either study. The time of participation inthe first study did not reflect experience in the absence of rifaximintherapy, therefore, for subjects who rolled over to the second studywithout an HE episode in the first study, the 12-month interval prior tothe second study was used for comparison. Most subjects in the secondstudy (152 of 266) were also in the first study. Overt HE episodes inthe second study were combined with the first study because, unlike thefirst study, subjects in the second study had the option of remaining onrifaximin after experiencing their first breakthrough HE episode. Thenumbers of overt HE episodes experienced during the 6-month or 12-monthintervals prior to the first study or prior to the second study wereknown. While 30.8% of subjects had >2 HE episodes during the 6-month or12-month interval prior to rifaximin therapy, only 3.6% of subjectshad >2 HE episodes during rifaximin therapy for up to 840 days (medianexposure=253 days [˜8 months]) in the first study plus the second study.This difference in the incidence of HE episodes while subjects werereceiving rifaximin when compared to the absence of rifaximin therapysuggests a strong effect of rifaximin in relieving the burden of overtHE episodes in patients with recurrent, overt HE associated severe liverdisease.

Hepatic encephalopathy is a serious, rare, complex, episodic,neuropsychiatric syndrome associated with advanced liver disease.Hepatic encephalopathy is a formidable burden on the patient, his/herfamily, and the healthcare system. Overt HE episodes are debilitating,render the patient incapable of self-care, and frequently result inhospitalization. Rifaximin has been granted orphan drug status for theHE indication because the disease is serious and chronicallydebilitating, and there is a low incidence of HE in the generalpopulation. Also, there is an unmet medical need for patients with HEbecause of limitations of the current standard of care.

Without wishing to be bound by any specific scientific theories, it isbelieved that the mechanism of action of rifaximin depends on theinhibition of DNA-dependent RNA polymerase of the target microorganisms,leading to the suppression of initiation of chain formation in RNAsynthesis. Rifaximin has a lower rate of fecal eradication of pathogenscompared with other commonly used antibacterial drugs and causes minimalalterations in gut flora suggesting that rifaximin has a differentmechanism of action than other commonly used drugs in enteric bacterialinfection, such as the fluoroquinolones. The antibacterial properties ofrifaximin appear to result from bactericidal activity at rifaximinconcentrations greater than or equal to the MIC, and from alterations inbacterial morphology and physiological functioning, which have beenobserved at sub-MIC concentrations.

It was unexpectedly discovered herein, that the risk of the developmentof antibiotic resistance is low during chronic treatment with rifaximinwhen compared to other systemic antibiotics such as neomycin. The lowrisk of antibiotic resistance during rifaximin therapy is likely due tothe fact that resistance to rifaximin is not plasmid-mediated butinstead requires a stable mutation in host cell DNA; therefore,dissemination of resistance and cross-resistance to other antibiotics byplasmid-based mechanisms are eliminated. Also, bacteria at sites outsideof the GI tract are not exposed to appreciable selective pressurebecause of negligible systemic concentrations of rifaximin.Additionally, microbiological data from a study of patients withulcerative colitis who were receiving high doses of rifaximin showedthat rifaximin-resistant bacterial colonies generated during in vivoexposure to rifaximin were unstable and susceptibility returned after abrief period of treatment interruption.

Rifaximin treatment results in fewer overt HE episodes that mayotherwise incapacitate the patient, may alleviate the burden on familymembers who are required to care for the patient, and reduces the burdenof hospitalization in this patient population and the healthcare system.The following are results from the second study with respect to time tofirst breakthrough overt HE episode:

The protective effect was reproducible: the time to first breakthroughovert HE episode results were similar between the rifaximin group in thefirst study and new rifaximin subjects in the second study; and 22% and27.6% had breakthrough overt HE in the first study rifaximin group andthe second study new rifaximin group, respectively. Adjusted forexposure, rates of breakthrough HE episodes were 0.62 events/PEY in therifaximin group from the first study compared to 0.38 events/PEY for newrifaximin subjects in the second study. These data demonstrate thatprotection against breakthrough overt HE in subjects who receivedrifaximin was consistent between the 2 studies. Additionally, when thefirst study placebo subjects crossed over to rifaximin therapy byentering the second study, a striking protective effect of rifaximin wasobserved in the comparison of Kaplan-Meier estimates of time to firstbreakthrough overt HE between placebo experience in the first study andrifaximin experience in the second study. The hazard ratio of rifaximinto placebo was 0.302 (95% CI: 0.166 to 0.549, p<0.0001 for between groupdifference in relative risk). This result represents 70% reduction inrisk of experiencing breakthrough overt HE during rifaximin treatment inthe second study when compared with their prior placebo experience inthe first study. This reduction took place in spite of the aging andpresumably progressing nature of the population with chronic liverdisease.

The protective effect was durable: the Kaplan-Meier estimate oftime-to-first breakthrough HE demonstrated long-term maintenance ofremission from breakthrough HE when rifaximin subjects in remissionafter participation in the first study were followed in the second study(up to 680 days of rifaximin therapy; median exposure durations were 168days in the first study and 253 days in the second study). The incidenceof breakthrough HE episode for these rifaximin subjects relative to thefirst study placebo was dramatically low, an indication of fewerbreakthrough HE episodes with rifaximin treatment (p<0.0001 fordifference in relative risk between rifaximin and placebo).

Results for other efficacy endpoints also demonstrated statisticallysignificant protective effects of rifaximin. In the first study , theanalysis of time to first HE-related hospitalization (e.g.,hospitalization directly resulting from HE or hospitalizationcomplicated by HE) demonstrated that the reduction in risk ofhospitalization due to HE was 50% in the rifaximin group, when comparedwith placebo, during the 6-month treatment period (p=0.0129 forbetween-group difference in relative risk). In the first study , therisk of HE-caused hospitalization (e.g., hospitalization directlyresulting from HE only) was reduced by 56% (p=0.0064 for between-groupdifference in relative risk), and the risk of all-cause hospitalizationwas reduced by 30% in the rifaximin group compared with the placebogroup (p=0.0793 for between-group difference in relative risk). In thefirst study, the risk of all-cause hospitalization rate was reduced by30% in the rifaximin group when compared to placebo (p=0.0793 forbetween-group difference in relative risk). The all-causehospitalization rate was 0.92 events/PEY in the rifaximin group versus1.31 event/PEY in the placebo group.

In the second study, the low HE-caused hospitalization rate wasmaintained at rates consistent with those in the first study: HE-causedhospitalization rate was 0.29 event/PEY and all cause hospitalization inthe second study was 0.66 event/PEY. The consistently lowHE-related/HE-caused hospitalization rate in rifaximin-treated subjectsin the first study and in the second study was at least partly a resultof maintaining remission from demonstrated HE in subjects with end-stageliver disease.

Improved Quality of Life in HE Subjects Administered Rifaximin

HE is manifested as a continuum of mental status deterioration,psychomotor dysfunction, impaired memory, increased reaction time,sensory abnormalities, poor concentration, disorientation, and in severeforms, coma. Patients with HE experience symptoms that have adverseconsequences for the patient's health-related quality of life, andresult in a decreased ability for self care. The Chronic Liver DiseaseQuestionnaire (CLDQ) is a validated instrument for measuringhealth-related quality of life in subjects with chronic liver disease.The mean change from baseline in CLDQ fatigue domain scores at end oftreatment was 1 of 5 key secondary endpoints in this study.Additionally, mean change from baseline in CLDQ scores (overall scoreand each domain score) at each postbaseline assessment and at end oftreatment was one of the tertiary efficacy endpoints prespecified in thestudy protocol.

The CLDQ was administered at Baseline, Days 28, 56, 84, 112, and 140,and at Day 168 or end of treatment. The CLDQ includes 29 items in thefollowing 6 domains: abdominal symptoms (three items), fatigue (fiveitems), systemic symptoms (five items), activity (three items),emotional function (eight items), and worry (five items). Scores in thefatigue subdomain were highly correlated with liver disease severity asdetermined by clinical assessments. Therefore, the fatigue subdomain waschosen as a key secondary endpoint for the study.

Subjects ranked their level of fatigue by using a 7-point scale from theworst response (1, high degree of fatigue) the best response (7, minimalfatigue). Other domains and the overall score were also ranked on a7-point scale with higher scores indicating better quality of life andlower scores reflecting lower quality of life. For example, 1 of the 5fatigue items was ‘How much time have you been fatigued during the last2 weeks?’ Response options were ‘all of the time,’ ‘most of the time,’‘a good bit of the time,’ ‘some of the time,’ ‘a little of the time,’‘hardly any of the time,’ and ‘none of the time.’ These were graded as 1(worst degree of fatigue), 2, 3, 4, 5, 6, and 7 (no fatigue),respectively.

In contrast to the change from baseline analysis, the AUC analysispresented below includes CLDQ results over the subject's complete timeof participation in the study.

Area Under the Curve and Time-Weighted Average (Twa) Analysis of ChronicLiver Disease Questionnaire Results

In the an original analysis of the study data, minimal differencesbetween placebo and rifaximin groups were observed in the changes frombaseline in CLDQ fatigue scores and in other CLDQ domain scores. Mean(SD) fatigue scores were 3.34 (1.406) versus 3.28 (1.326) at baselineand 3.51 (1.529) versus 3.57 (1.527) at last assessment in the placeboand rifaximin groups, respectively.

In this example, CLDQ responses were tracked over time for each subjectand an area under the curve (AUC) was determined. For those subjects whohad breakthrough HE episodes, CLDQ data reflect experience prior to thebreakthrough episode. Because all subjects did not stay in the study forthe same length of time, the AUC was normalized by exposure time (T),referred to as Time-weighted average (Twa) as given below:

${{Twa} = \frac{AUC}{T}},$

Thus, Twa describes the average CLDQ response from baseline through thecourse of the trial, normalized to duration of exposure.

As shown below in FIG. 14A-B, there is a distinct separation in CLDQresults, as measured by Twa, between the rifaximin and placebo groups inthe frequency distributions of Twa scores for the fatigue domain andoverall domain. The shift in the frequency distribution toward higherscores in the rifaximin subjects indicates better responses; ie,improved overall quality of life results and less fatigue for therifaximin group when compared with the placebo group. Similarbetween-group differences favoring the rifaximin group were observed inthe frequency distributions for additional CLDQ domains.

Table 25 summarizes Twa results for the CLDQ overall domain scores andCLDQ fatigue domain scores by treatment group in the Intent-to-Treat(ITT) population. Time-weighted average scores for the fatigue domainand overall domain were significantly higher in the rifaximin group thanin the placebo group (p=0.0087 [fatigue domain] and p=0.0093 [overalldomain] for between-group differences in favor of the rifaximin group).Time-weighted average scores for other CLDQ domains were alsosignificantly higher in the rifaximin group than in the placebo group(p=0.0090 [abdominal symptoms], p=0.0160 [systemic symptoms], p=0.0022[activity], p=0.0065 [emotional function], and p=0.0436 [worry] forbetween-group differences in favor of the rifaximin group.

The differences in mean Twa scores between treatment groups (rifaximinminus placebo) were 0.72 for the fatigue domain and 0.75 for the overalldomain. A difference of 0.5 points is considered important and adifference of 0.8 points is considered ‘large’ when using a 7-pointscale for quality-of-life measurements.

Pertinent findings from the literature comparing subjects with nocirrhosis, Child-Pugh A, and Child-Pugh C liver disease suggest thatdifferences of 0.5 to 1.0 in CLDQ scores are clinically significant.Mean differences in overall domain scores in subjects with no cirrhosiscompared with subjects with Child-Pugh A (no cirrhosis minus Child-PughA) were 0.6 in subjects in the United States and 0.3 in a study ofsubjects in Spain. Greater mean differences in overall domain scoreswere reported for the transition from no cirrhosis to Child-Pugh C (nocirrhosis minus Child-Pugh C): 1.3 difference in the United States studyand 1.0 difference in the Spain study.

TABLE 25 Area Under the Curve and Time-Weighted Average for CLDQ Results(ITT Population) CLDQ results Rifaximin Placebo P- parameter N = 140 N =159 value^(a) Fatigue domain Baseline score n = 81 n = 86 Mean (SD) 3.37(1.304) 3.46 (1.363) Median (min,  3.40 (1.0, 5.8)  3.40 (1.4, 6.4) max)Twa (score) n = 82 n = 86 Mean (SD) 3.242 (1.7619) 2.522 (1.7538) p =0.0087 Median (min,    3.600 (0.29, 6.51)    2.365 (0.27, 6.25) max)Overall domain Baseline score n = 82 n = 87 Mean (SD) 4.18 (1.184) 4.31(1.058) Median (min,  4.25 (1.6, 6.7)  4.29 (1.7, 6.6) max) Twa (score)n = 83 n = 87 Mean (SD) 3.692 (1.8607) 2.943 (1.8480) p = 0.0093 Median(min,    4.249 (0.39, 6.70)    2.926 (0.46, 6.63) max)

Conclusions for CLDQ Analyses

When CLDQ results from the study were analyzed over the duration ofexposure to study drug by calculation of Twa, subjects in the rifaximingroup had significantly less fatigue and significantly greater overallquality of life than subjects in the placebo group. For example, mean(±SD) Twa fatigue scores were 3.24 (1.76) in the rifaximin group and2.42 (1.75) in the placebo group (p=0.0087 in favor of the rifaximingroup). Significant differences in Twa CLDQ results in favor of therifaximin group were also observed for the overall CLDQ domain score(p=0.0093), and for each of the other component domains of the CLDQ,including abdominal symptoms (p=0.0090), systemic symptoms (p=0.0160),activity (p=0.0022), emotional function (p=0.0065), and worry(p=0.0436).

Importantly, these data demonstrate that rifaximin treatment resulted insignificantly improved quality of life compared to placebo over a6-month treatment period in subjects with hepatic cirrhosis andrecurrent, overt HE, prior to experiencing a new onset overt HE andperformed without data impugnation. This demonstrates that the patientsin this study reported improvement in every domain relative to theplacebo group. The observed statistically significant difference betweenthe rifaximin and placebo groups agrees with the clinically significantdifferences reported for subjects with increasing severity of liverdisease as measured by Child-Pugh score.

Correlation of CFF to Breakthrough Overt HE

As a test of the reliability and clinical relevance of the primaryendpoint, the quantitative results for CFF were tested for correlationto the occurrence of breakthrough overt HE (primary efficacy measure),which was determined on the basis of clinical symptoms using Conn score(or a combination of Conn score and asterixis grade).

The CFF values were tracked over time for each subject and it was notedthat on average, subjects who experienced a breakthrough HE had lowertest values than subjects who did not experience a breakthrough event.And it was further noted that the area under the CFF versus time curve(AUC) could be used to accurately describe the variation in the CFF overtime for each subject as a Time-weighted average (twa). Since allsubjects did not stay in the study for the same length of time, the twawas normalized by exposure time (T).

The results of the CFF test over time were expressed as:

${{twa} = \frac{AUC}{T}},$

-   -   where T is the exposure time. Thus, twa describes the average        CFF effect through the trial.

The correlation between twa and the presence or absence of breakthroughHE episode was analyzed with analysis of variance and Spearman rankcorrelation coefficient. Additionally, a ROC curve analysis wasperformed to evaluate the accuracy of the twa to discriminate betweenthe presence or absence of breakthrough episodes. In a ROC curve, thetrue positive rate (Sensitivity) is plotted against the false positiverate (1-Specificity). A diagnostic test with perfect discrimination hasa ROC plot that passes through the upper left corner (100% sensitivity,100% specificity). Therefore the closer the ROC plot to the upper leftcorner, the higher the overall accuracy of the test.

FIGS. 31 and 32 and Table 26 demonstrate that the difference between thefrequency distributions of twa corresponding to the presence (mean=12.5Hz) and absence of breakthrough HE events (mean=32.7 Hz) wasstatistically significant (p<0.0001). Also, mean twa correlated withpresence or absence of breakthrough HE episode (Spearman correlationcoefficient=−0.62; p<0.0001).

TABLE 26 Area Under the Curve and Time-Weighted Average for CFF Results(ITT Population) Non-breakthrough HE Breakthrough HE CFF resultsparameter N = 195 N = 104 P-Value^(a) AUC₍₁₋₁₆₈ _(days))(Hz × day) n =194 n = 99 Mean (SD) 5455.07 (1918.260) 2090.24 (1648.022)     Median(min, max)     6037.0 (137.4, 8189.4)  1610.2 (175.7, 7092.9) twa(HZ)^(b) n = 194 n = 99 Mean (SD) 32.67 (11.487) 12.52 (9.868)     p <0.0001 Median (min, max)    36.2 (0.8, 49.0) 9.6 (1.1, 42.5) CFF:critical flicker frequency; AUC: area under the ammonia concentrationversus time curve; twa: time-weighted average ^(a)p-value calculatedusing ANCOVA model with effects for treatment and analysis region ascovariates. ^(b)Spearman's correlation for twa to presence or absence ofbreakthrough HE equals −0.62; p < 0.0001

The ROC curve analysis of twa for the diagnosis of breakthrough HE byCFF showed an area under the curve value of 0.88 (95% CI 0.84-0.92).Values close to 1, and the appearance of the ROC plot closer to theupper left corner, are considered diagnostically significant.

Thus, CFF, which is an accepted, physiologically relevant, quantitativemeasure associated with HE, was shown to be highly predictive ofbreakthrough HE as defined by as an increase of Conn score to Grade≥2(ie, 0 or 1 to ≥2) or an increase in Conn and asterixis score of 1 gradeeach for those subjects who entered the study with a Conn score of 0.The fact that this quantitative measure discriminates in a highlystatistically significant manner demonstrates the reliability andclinical relevance of the primary efficacy measure.

Example 6: Correlation of the Venous Ammonia Levels to HE BreakthroughEvents

Subjects administered rifaximin had significantly greater reductions invenous ammonia levels when compared to placebo-treated subjects(p=0.0391, see Table 27). Venous ammonia levels were assessed atScreening, Baseline, Day 28, Day 84, and Day 168/end-of-treatment.

TABLE 27 Mean (SD) Changes from Baseline in Venous Ammonia Level byTreatment Group (ITT Population) Placebo Rifaximin N = 159 N = 140 P-(μg/dL) (μg/dL) Value^(a) Baseline n = 146 n = 132 Mean (SD) ammonialevel 90.3 (52.48) 87.9 (47.76) End of treatment n = 141 n = 132 Mean(SD) ammonia level 88.4 (45.75) 83.9 (45.02) Change from baseline to endn = 131 n = 125 of treatment Mean (SD) change in ammonia −0.3 (58.13)−5.7 (46.77) p = level 0.0391 Note: Baseline value was the lastavailable value prior to first dose of study drug, and end of treatmentvalue was the last available postbaseline value during the treatmentperiod. ^(a)P-value was calculated using analysis of covariance witheffects for treatment and analysis region, and baseline as a covariate.

Blood ammonia levels, a quantitative assessment that is associated withthe CNS effects underlying overt HE, was found to be highly correlatedto the occurrence of breakthrough overt HE as determined by clinicalevaluation. This high degree of correlation was consistent with thecorrelation between CFF results and the occurrence of breakthrough overtHE.

The venous ammonia laboratory values were tracked over time for eachsubject. To normalize by exposure time, a twa value was calculatedsimilarly to the CFF analysis.

The correlation between the ammonia twa and the presence or absence ofbreakthrough HE episode was analyzed as described for the CFF.

FIG. 33 and Table 28 show that the difference between the frequencydistributions of twa corresponding to the presence (mean=102.4 mol/L)and absence of breakthrough HE events (mean=85.4 mol/L) wasstatistically significant (p=0.0079). Also, mean twa correlated withpresence or absence of breakthrough HE episode (Spearman correlationcoefficient of 0.22, p=0.0005).

TABLE 28 Area Under the Curve and Time-Weighted Average for VenousAmmonia Concentrations (ITT Population) Venous ammonia Non-breakthroughHE Breakthrough HE concentration parameter N = 195 N = 104 P-Value^(a)AUC_((1-28 days))(μmol/L × day) n = 173 n = 68 Mean (SD) 2304.83(1211.428)     2763.93 (1160.357)    Median (min, max) 2038.5 (499.5,9153.0)  2787.8 (999.0, 7681.5) twa (μmol/L)^(b) n = 173 n = 68 p =0.0079 Mean (SD) 85.36 (44.87)      102.37 (42.98)     Median (min, max)75.5 (18.5, 339.0) 103.25 (37.0, 284.5) AUC: area under the ammoniaconcentration versus time curve; twa: time-weighted average. ^(a)p-valuecalculated using ANCOVA model with effects for treatment and analysisregion as covariates. ^(b)Spearman's correlation for twa to presence orabsence of breakthrough HE equals 0.22; p = 0.0005

The ROC curve analysis of twa for the diagnosis of breakthrough HE byvenous ammonia levels, showed an area under the curve value of 0.64 (95%CI 0.57-0.72) (See FIG. 34 ). Values close to 1, and the appearance ofthe ROC plot closer to the upper left corner, are considereddiagnostically significant.

Thus, venous ammonia level, which is an accepted physiologicallyrelevant quantitative measure associated with HE, was shown to be highlypredictive of breakthrough HE as defined by as an increase of Conn scoreto Grade≥2 (ie, 0 or 1 to ≥2) or an increase in Conn and asterixis scoreof 1 grade each for those subjects who entered the study with a Connscore of 0. The fact that this quantitative measure discriminates in ahighly statistically significant manner the presence or absence ofbreakthrough HE attests to the reliability and clinical relevance of theprimary efficacy measure.

INCORPORATION BY REFERENCE

The contents of all references, patents, pending patent applications andpublished patents, cited throughout this application are herebyexpressly incorporated by reference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A method of reducing the risk of hospitalization for a subject havinghepatic encephalopathy (HE) comprising administering between about 1000mg to about 1200 mg rifaximin daily to the subject for a period of 12months or longer, wherein the subject has a Model for End-Stage LiverDisease (MELD) score of between about 1 to 24, thereby reducing the riskof hospitalization.
 2. The method of claim 1, wherein the amount ofrifaximin is sufficient to reduce the frequency of hospitalizationvisits by at least 50 percent compared to a subject who is not beingadministered rifaximin.
 3. The method of claim 1, wherein the subjecthas a MELD score of less than or equal to
 10. 4. The method of claim 1,wherein the subject has a MELD score of between 11 and
 18. 5. The methodof claim 1, wherein the subject has a MELD score of between 19 and 24.6. The method of claim 1, wherein the HE is overt hepaticencephalopathy.
 7. The method of claim 1, wherein the rifaximin isadministered orally.
 8. The method of claim 1, wherein 1100 mg ofrifaximin is administered per day.
 9. The method of claims 1, wherein550 mg of rifaximin is administered twice daily.
 10. The method of claim1, further comprising administering lactulose.